U.S. patent application number 14/906970 was filed with the patent office on 2016-06-23 for system and method for processing fluid in a fluidic cartridge.
The applicant listed for this patent is ATLAS GENETICS LIMITD. Invention is credited to Ben Arlett, Jay Kendall Taylor.
Application Number | 20160175836 14/906970 |
Document ID | / |
Family ID | 49167112 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160175836 |
Kind Code |
A1 |
Taylor; Jay Kendall ; et
al. |
June 23, 2016 |
System and Method for Processing Fluid in a Fluidic Cartridge
Abstract
A system and method for processing fluid in a fluidic cartridge
is provided. The system comprises a fluid pathway for passing a
liquid sample therethrough from an upstream end to a downstream
end, a sample processing chamber within the fluid pathway having an
inlet valve upstream of the sample processing chamber, a downstream
sample processing region within the fluid pathway downstream of the
outlet valve and a bypass channel coupled to the fluid pathway at a
junction between the outlet valve and the downstream sample
processing region, the valve system configured such that surplus
liquid downstream of the outlet valve may be evacuated through the
bypass channel when the outlet valve is closed, thereby leaving a
metered volume of liquid sample between the inlet valve and the
downstream sample processing region.
Inventors: |
Taylor; Jay Kendall;
(Ottawa, CA) ; Arlett; Ben; (Bristol, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ATLAS GENETICS LIMITD |
Thowbridge Wiltshire |
|
GB |
|
|
Family ID: |
49167112 |
Appl. No.: |
14/906970 |
Filed: |
July 28, 2014 |
PCT Filed: |
July 28, 2014 |
PCT NO: |
PCT/GB2014/052307 |
371 Date: |
January 22, 2016 |
Current U.S.
Class: |
435/287.2 ;
422/516 |
Current CPC
Class: |
B01L 2300/0816 20130101;
B01L 3/50273 20130101; B01L 3/502738 20130101; C12Q 1/6844
20130101; B01L 2400/0487 20130101; B01L 2400/0622 20130101; B01L
2200/0605 20130101; G01N 35/1097 20130101; B01L 2400/0633 20130101;
B01L 2300/12 20130101; B01L 2300/0861 20130101; B01L 2400/0655
20130101; B01L 3/502 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12Q 1/68 20060101 C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 29, 2013 |
GB |
1313524.9 |
Claims
1. A valve system in a fluidic cartridge for metering a liquid
sample in a sample processing region, comprising: a fluid pathway
for passing a liquid sample therethrough from an upstream end to a
downstream end; a sample processing chamber within the fluid
pathway having an inlet valve upstream of the sample processing
chamber and an outlet valve downstream of the sample processing
chamber; a downstream sample processing region within the fluid
pathway downstream of the outlet valve; and a bypass channel
coupled to the fluid pathway at a junction between the outlet valve
and the downstream sample processing region, the valve system
configured such that surplus liquid sample downstream of the outlet
valve may be evacuated through the bypass channel when the outlet
valve is closed, thereby leaving a metered volume of liquid sample
in the fluid pathway between the inlet valve and the downstream
sample processing region.
2. The valve system of claim 1, wherein the downstream sample
processing region comprises a target chamber.
3. The valve system of claim 1 or 2, further comprising: a
plurality of fluid pathways, each for passing a liquid sample
through from an upstream end to a downstream end; a sample
processing chamber within each fluid pathway and each having an
inlet valve upstream of the sample processing chamber and an outlet
valve downstream of the sample processing chamber; a downstream
sample processing region within each fluid pathway downstream of
the respective outlet valve; and a bypass channel coupled to each
fluid pathway at a junction between the downstream sample
processing region and the outlet valve therein, the valve system
configured such that surplus liquid sample downstream of the outlet
valve may be evacuated through the respective bypass channel when
the outlet valve is closed, thereby leaving a plurality of metered
volumes of liquid sample in the plurality of fluid pathways between
the respective inlet valve and the respective downstream sample
processing regions.
4. The valve system of any preceding claim, wherein the or each
fluid pathway further comprises at least one compressible element
downstream of the at least one downstream sample processing
regions, the at least one compressible element configured to become
increasingly biased against fluid upstream of the compressible
element as the liquid sample passes through the open outlet valve
so as to increase the pressure in the fluid pathway, such that
surplus liquid sample downstream of the outlet valve may be
expelled from the fluid pathway and into the bypass channel by the
at least one compressible element when the outlet valve is closed
and whilst the pressure in the bypass channel is less than the
pressure in the fluid pathway.
5. A valve system in a fluidic cartridge for expelling liquid
sample from a sample processing region, the apparatus comprising: a
fluid pathway for passing a liquid sample therethrough from an
upstream end to a downstream end; an outlet valve within the fluid
pathway, the outlet valve configured to move between a closed
position in which it prevents the liquid sample from passing
through the outlet valve and an open position in which it permits
the liquid sample to pass through the outlet valve; a downstream
sample processing region within the fluid pathway downstream of the
outlet valve; a bypass channel coupled to the fluid pathway at a
junction between the outlet valve and the downstream sample
processing region, the valve system configured such that liquid
sample downstream of the outlet valve may be expelled through the
bypass channel when the outlet valve is in its closed position; and
at least one compressible element downstream of the downstream
sample processing region, the at least one compressible element
configured to become increasingly biased against fluid upstream of
the compressible element as the liquid sample passes through the
open outlet valve, such that the liquid sample downstream of the
outlet valve may be expelled from the fluid pathway and into the
bypass channel by the at least one compressible element when the
outlet valve is closed.
6. The valve system of claim 5, further comprising a sample
processing chamber within the fluid pathway and upstream of the
outlet valve.
7. The valve system of any one of claims 4 to 6, wherein the at
least one compressible element is a gas spring comprising a blind
bore filled with a compressible fluid.
8. The valve system of any preceding claim, further comprising a
bypass valve located within the or each bypass channel, the bypass
valve configured to move between a closed position in which it
prevents the liquid sample from passing through the bypass valve
and an open position in which it permits the liquid sample to pass
through the bypass valve.
9. The valve system of any preceding claim, wherein at least one of
the valves in the valve system is a pneumatically-actuated
valve.
10. The valve system of claim 9, wherein the at least one
pneumatically-actuated valve comprises a valve chamber having first
and second openings connected to the pathway or channel,
respectively; and a flexible membrane movable between a closed
position, in which the flexible membrane seals against the first
and second openings to prevent fluid flow through the pathway or
channel, and an open position, in which the flexible membrane is
spaced apart from the first and second openings to permit fluid to
flow through the pathway or channel.
11. The valve system of claim 10, further comprising a pneumatic
interface for connecting to a source of positive and/or gauge gas
pressure, the pneumatic interface comprising a plurality of
ports.
12. The valve system of claim 11, wherein the or each valve further
comprises a fluid passageway having an opening in the valve
chamber, the opening separated from the first and second openings
by the flexible membrane, wherein the fluid passageway is coupled
to a port in the pneumatic interface for applying a positive or
negative gas pressure in the valve chamber to move the flexible
membrane between the open and closed positions.
13. The valve system of any one of claims 9 to 12, when dependent
on any one of claims 1 to 3, wherein the inlet and outlet valves
are configured to be actuated simultaneously.
14. The valve system of any preceding claim, wherein the or each
bypass channel is connected to the fluid pathway immediately
downstream of the outlet valve to as to minimise or eradicate a
deadleg between the outlet valve and the bypass channel.
15. The valve system of any one of claims 1 to 4 and 6 to 13,
wherein the sample processing chamber is a nucleic acid
amplification chamber; wherein the downstream sample processing
region is a detection chamber; and wherein ratio of detection
chambers to nucleic acid amplification chambers is 2:1.
16. The valve system of any one of claims 4 to 14, wherein each
downstream sample processing region is coupled to a single
compressible element.
17. The valve system of claim 10, wherein the valve chamber is
formed in a first polymer layer, preferably a pneumatic layer of
the fluidic cartridge.
18. The valve system of claim 17, when dependent on claim 11,
wherein the pneumatic interface is formed in the first polymer
layer.
19. The valve system of any preceding claim, wherein the fluid
pathway is formed in a second polymer layer, preferably a fluidic
layer of the fluidic cartridge.
20. The valve system of claim 19, wherein the bypass channel is
formed in the second polymer layer.
21. The valve system of claim 10, wherein the valve membrane
comprises a thermoplastic elastomer.
22. The valve system of claim 18, wherein the first polymer layer
comprises polypropylene.
23. The valve system of claim 19 or 20, wherein the second polymer
layer comprises polypropylene.
24. A method of metering a liquid sample in a fluidic cartridge
comprising a fluid pathway having a sample processing chamber
therein, an inlet valve upstream of the sample processing chamber
and an outlet valve downstream of the sample processing chamber, a
downstream sample processing region therein, and a bypass channel
coupled to the fluid pathway at a junction between the outlet valve
and the downstream sample processing region; the method comprising:
passing a liquid sample through the inlet valve, into the first
chamber, and through the outlet valve; closing the outlet valve and
evacuating surplus liquid sample downstream of the outlet valve
through the bypass channel to empty the fluid pathway downstream of
the outlet valve of fluid, thereby leaving a metered volume of
liquid sample in the fluid pathway between the inlet valve and the
downstream sample processing region; and opening the outlet valve
and delivering the metered volume of liquid sample to the
downstream sample processing region.
25. The method of claim 24 wherein the fluidic cartridge further
comprises at least one compressible element downstream of the
downstream sample processing region, wherein the step of passing a
liquid sample through the inlet valve, into the first chamber, and
through the outlet valve further comprises compressing the
compressible element as the liquid sample passes downstream of the
outlet valve; and wherein the step of evacuating surplus liquid
sample downstream of the outlet valve further comprises the
compressible element exerting a force against the surplus liquid
sample to expel it from the fluid pathway and into the bypass
channel.
26. The method of claim 24 or claim 25, wherein the fluidic
cartridge further comprises a bypass valve in the bypass channel,
and wherein the method further comprises: closing the bypass valve
prior to the step of passing a liquid sample through the inlet
valve, into the first chamber, and through the outlet valve; and
wherein the step of evacuating surplus liquid sample downstream of
the outlet valve further comprises opening the bypass valve.
27. The method of any one of claims 24 and 25, wherein the step of
closing the inlet and outlet valves comprises closing the inlet and
outlet valves simultaneously.
28. A method of expelling surplus liquid sample from a fluidic
cartridge comprising a fluid pathway having a downstream sample
processing region therein, an outlet valve upstream of the
downstream sample processing region, a bypass channel coupled to
the fluid pathway at a junction between the outlet valve and the
downstream sample processing region, and a compressible element
downstream of the downstream sample processing region; the method
comprising: passing a liquid sample through the outlet valve,
thereby compressing the compressible element as the liquid sample
passes downstream of the outlet valve; and closing the outlet valve
and evacuating surplus liquid sample downstream of the outlet valve
through the bypass channel by the compressible element exerting a
force against the surplus liquid sample to expel it from the fluid
pathway and into the bypass channel.
29. The method of claim 28, wherein the fluidic cartridge further
comprises a bypass valve in the bypass channel, and wherein the
method further comprises: closing the bypass valve prior to the
step of passing a liquid sample through the outlet valve; and
wherein the step of evacuating surplus liquid sample downstream of
the outlet valve further comprises opening the bypass valve.
Description
FIELD
[0001] The present invention relates to valve systems and
corresponding methods for processing a liquid sample in a fluidic
cartridge. More specifically, the present invention relates to a
metering system for delivering a volume of liquid sample and a
system for expelling excess liquid sample.
BACKGROUND
[0002] Sample preparation and analysis presents many logistical
problems. Conventionally, many medical samples (such as blood,
saliva, urine and swab eluate) are provided to a doctor, for
example a general practitioner doctor (GP) or a principle care
physician (PCP), in a local surgery without the equipment necessary
to analyse the sample. Hence, the sample must be sent to a
laboratory where the sample is analysed. The test results must then
be collated and returned to the GP to analyse the results and make
a diagnosis. This approach is inadequate. Firstly, there is a
significant risk that a sample is lost in transit or mismatched
with the wrong patient. Moreover, whilst recent developments in
technology have reduced the overall time taken to conduct the test,
the delay involved in sending the sample to a laboratory is
unsatisfactory.
[0003] Nevertheless, analytical systems of the kind found in
laboratories are complex and it is often difficult to provide
sufficient amounts of pure targets from source samples to reliably
perform downstream analytical assays. This typically prohibits
local GP surgeries from being able to carry out such tests on
site.
[0004] However, in recent years efforts have been made to reduce
the scale of the analytical systems to make tests faster and
simpler to run, and require smaller quantities of sample. For
instance, "laboratory on a chip" (LOC) devices (a subset of
microfluidic devices) integrate almost all medical tests or
diagnostic operations performed in a hospital on a single
microfluidic chip. The channels forming such microfluidics devices
handle small fluid volumes and are connected together so as to
achieve a desired function such as mixing of a sample, moving the
sample through the device, reacting the sample with different
reagents, and so on. These chips may be inserted into machines to
control the performance of a test and measure the results.
[0005] However, it has been found that handling a sample in a
microfluidics device can be very difficult. In such small channels
as are found on a conventional LOC, it is difficult to apply
external forces to move the sample from one site to another to
perform different actions on the sample. There is also a limit to
the complexity of a LOC device which operates purely using
capillary action. Furthermore, owing to the small sample sizes of
LOC's, the devices have reduced sensitivity and the probability of
a target being present in the sample is thus reduced.
[0006] An alternative approach is to use a fluidic cartridge. The
scale of the components of a fluidic cartridge is larger than for a
microfluidic device, and so it becomes possible to move a sample
through various different sites to perform different actions on it.
This makes it possible to perform more complex tests than may be
conducted using typical LOC devices, whilst still providing an
analytical system of potential use in a local GP surgery.
[0007] Scientific assays useful in medical diagnostics have
increasingly involved biochemical procedures, such as the
polymerase chain reaction ("PCR"). The PCR assay has provided a
powerful method of assaying for the presence of defined segments of
nucleic acids. It is therefore desirable to perform a PCR assay on
a fluidic cartridge.
[0008] Reducing PCR to the microchip level is important for
portable detection technologies and high-throughput analytical
systems. The method can be used to assay body fluids for the
presence of nucleic acid specific for particular pathogens, such as
the Chlamydia trachomatis bacterium, HIV or any other pathogenic
microbe.
[0009] The introduction of commercially available automated DNA
amplification assays has allowed more laboratories to introduce
these technologies for routine testing of specimens. However, there
is a need to improve the fluidic devices used for this purpose.
[0010] It is requirement of devices employing PCR technology in the
analysis and processing of fluid samples that a well-defined volume
of processed fluid may be delivered to detection chambers for
analysis of the sample. It is particularly important in LOC type
cartridges where multiple fluid sample preparation steps are
performed within the cartridge that the tolerances of the channel
and chambers in the sample processing region are not allowed to
stack up prior to processing and analysis of the fluid sample as
this may lead to fairly large errors in the calculated amount of
fluid required to fill the PCR chamber and subsequently be
delivered to the detection chambers.
[0011] In certain LOC type cartridges, reagents may be dried down
in the PCR chamber. This means that over-filling the PCR chamber
and allowing an excessive amount of fluid sample to flow past the
sample processing region may lead to a lack of reagents remaining
in the PCR chamber. Under-filling the PCR chamber by even a small
amount may lead to air pockets in the PCR chamber which in turn may
lead to unstable thermocycling. To ensure that the PCR chamber is
completely filled, slight over-filling of the chamber may be
desired. However, even slightly overfilling the PCR chamber leads
to unprocessed fluid downstream of the PCR chamber which may then
dilute processed fluid sample as it is moved from the PCR chamber
to the detection chambers.
[0012] WO2009108260 discloses a method for delivering a fixed
volume of fluid to a microfluidic device comprising configuring a
device with a sample loop comprising a desired volume, wherein the
sample loop is removable, using one or more pneumatically actuated
valves on a microfluidic device to fill the sample loop with the
fixed volume of the fluid and delivering the fluid to the
microfluidic device. Thus, a specific volume of fluid in the sample
loop can be injected into the main channel by closing the
intermediate valve, opening the flow through valves (606 and 608)
and applying pressure to the main channel. The sample loop and a
pass-through microfluidic channel are fluidically connected at a
first junction and a second junction, and wherein at least one
junction comprises a T-valve.
[0013] GB200711618 discloses a process for determining the
concentration of nucleic acids in a sample in a microfluidic
device. The process includes introducing a sample into a first
chamber, carrying out a number of cycles of an amplification
reaction to be carried out in cycles for amplifying nucleic acids,
transferring a defined volume which is a fraction of the volume of
the first chamber, and which has amplified nucleic acids into a
second chamber and replacing the transferred defined volume with
fresh reagents for the amplification reactions. The volume transfer
means may be a controllable metering pump, a reciprocating pump or
a similar means which can be used to move a defined volume from the
first chamber to the second chamber.
SUMMARY OF INVENTION
[0014] In a first aspect of the present invention, there is
provided a valve system in a fluidic cartridge for metering a
liquid sample in a sample processing region, comprising: a fluid
pathway for passing a liquid sample therethrough from an upstream
end to a downstream end; a sample processing chamber within the
fluid pathway having an inlet valve upstream of the sample
processing chamber and an outlet valve downstream of the sample
processing chamber; a downstream sample processing region within
the fluid pathway downstream of the outlet valve; and a bypass
channel coupled to the fluid pathway at a junction between the
outlet valve and the downstream sample processing region, the valve
system configured such that surplus liquid sample downstream of the
outlet valve may be evacuated through the bypass channel when the
outlet valve is closed, thereby leaving a metered volume of liquid
sample in the fluid pathway between the inlet valve and the
downstream sample processing region. As will be appreciated, the
present invention allows metering and processing of a sample to
take place whilst the sample is held in the same position in the
cartridge (i.e. the sample processing region).
[0015] Preferably, the sample processing region of the present
invention comprises one or more PCR chambers.
[0016] Preferably, the downstream sample processing region
comprises a target chamber. The target chamber may be a detection
chamber containing electrodes for detecting analyte in a
sample.
[0017] The valve system may further comprise: a plurality of fluid
pathways, each for passing a liquid sample through from an upstream
end to a downstream end; a sample processing chamber within each
fluid pathway and each having an inlet valve upstream of the sample
processing chamber and an outlet valve downstream of the sample
processing chamber; a downstream sample processing region within
each fluid pathway downstream of the respective outlet valve; and a
bypass channel coupled to each fluid pathway at a junction between
the downstream sample processing region and the outlet valve
therein, the valve system configured such that surplus liquid
sample downstream of the outlet valve may be evacuated through the
respective bypass channel when the outlet valve is closed, thereby
leaving a plurality of metered volumes of liquid sample in the
plurality of fluid pathways between the respective inlet valve and
the respective downstream sample processing regions. By providing a
plurality of pathways, it is possible to split a prepared sample
into multiple sample processing chambers. Providing multiple sample
processing chambers increases the reliability of results when the
fluidic cartridge is used for detecting analyte in a sample.
[0018] The or each fluid pathway further may comprise at least one
compressible element downstream of the at least one downstream
sample processing regions, the at least one compressible element
configured to become increasingly biased against fluid upstream of
the compressible element as the liquid sample passes through the
open outlet valve so as to increase the pressure in the fluid
pathway, such that surplus liquid sample downstream of the outlet
valve may be expelled from the fluid pathway and into the bypass
channel by the at least one compressible element when the outlet
valve is closed and whilst the pressure in the bypass channel is
less than the pressure in the fluid pathway. The compressible
element utilises the pressure created as fluid is advanced along
the fluid pathway to expel surplus fluid in the bypass channel,
eliminating the need for additional components in the fluidic
cartridge for the purpose of expelling surplus fluid.
[0019] In a second aspect of the present invention there is
provided a valve system in a fluidic cartridge for expelling liquid
sample from a sample processing region, the apparatus comprising: a
fluid pathway for passing a liquid sample therethrough from an
upstream end to a downstream end; an outlet valve within the fluid
pathway, the outlet valve configured to move between a closed
position in which it prevents the liquid sample from passing
through the outlet valve and an open position in which it permits
the liquid sample to pass through the outlet valve; a downstream
sample processing region within the fluid pathway downstream of the
outlet valve; a bypass channel coupled to the fluid pathway at a
junction between the outlet valve and the downstream sample
processing region, the valve system configured such that liquid
sample downstream of the outlet valve may be expelled through the
bypass channel when the outlet valve is in its closed position; and
at least one compressible element downstream of the downstream
sample processing region, the at least one compressible element
configured to become increasingly biased against fluid upstream of
the compressible element as the liquid sample passes through the
open outlet valve, such that the liquid sample downstream of the
outlet valve may be expelled from the fluid pathway and into the
bypass channel by the at least one compressible element when the
outlet valve is closed. The compressible element utilises the
pressure created as fluid is advanced along the fluid pathway to
expel surplus fluid in the bypass channel, eliminating the need for
additional components in the fluidic cartridge for the purpose of
expelling surplus fluid.
[0020] The valve system may further comprise a sample processing
chamber within the fluid pathway and upstream of the outlet
valve.
[0021] The at least one compressible element may be a gas spring
comprising a blind bore filled with a compressible fluid. By
providing a gas spring comprising a blind bore, a compressible
element may be provided without significantly complicating the
manufacturing process or increasing costs.
[0022] The valve system may further comprise a bypass valve located
within the or each bypass channel, the bypass valve may be
configured to move between a closed position in which it prevents
the liquid sample from passing through the bypass valve and an open
position in which it permits the liquid sample to pass through the
bypass valve. This allows control over when the fluid is removed
from the bypass channel.
[0023] At least one of the valves in the valve system may be a
pneumatically-actuated valve.
[0024] The at least one pneumatically-actuated valve may comprise a
valve chamber having first and second openings connected to the
pathway or channel, respectively; and a flexible membrane movable
between a closed position, in which the flexible membrane seals
against the first and second openings to prevent fluid flow through
the pathway or channel, and an open position, in which the flexible
membrane is spaced apart from the first and second openings to
permit fluid to flow through the pathway or channel.
[0025] The valve system may further comprise a pneumatic interface
for connecting to a source of positive and/or gauge gas pressure,
the pneumatic interface comprising a plurality of ports.
[0026] The or each valve may further comprise a fluid passageway
having an opening in the valve chamber, the opening separated from
the first and second openings by the flexible membrane, wherein the
fluid passageway is coupled to a port in the pneumatic interface
for applying a positive or negative gas pressure in the valve
chamber to move the flexible membrane between the open and closed
positions. Preferably the valve membrane is biased in the closed
position and a negative or gauge pressure is applied to move the
valve membrane from the closed to the open position. By providing
valves which are biased in the closed position, the valves are
biased against leakage and prevent leakage during temporary power
loss.
[0027] The inlet and outlet valves may be configured to be actuated
simultaneously. This avoids inadvertently pressurising the sample
processing chamber as the inlet and outlet valves are closed and
opened.
[0028] The or each bypass channel may be connected to the fluid
pathway immediately downstream of the outlet valve to as to
minimise or eradicate a deadleg between the outlet valve and the
bypass channel. This is advantageous because it minimises or
eradicates the surplus downstream of the sample processing chamber
which may dilute the processed sample as it moves along the fluid
pathway.
[0029] The sample processing chamber may be a nucleic acid
amplification chamber; wherein the downstream sample processing
region is a detection chamber; and wherein ratio of detection
chambers to nucleic acid amplification chambers is 2:1. Providing a
2:1 ratio facilitates duplex amplification of the sample.
[0030] Each downstream sample processing region may be coupled to a
single compressible element. Therefore each fluid pathway is
provided with means for expelling surplus fluid from the bypass
channel.
[0031] The valve chamber may be formed in a first polymer layer,
preferably a pneumatic layer of the fluidic cartridge. The
pneumatic interface may be formed in the first polymer layer. The
fluid pathway may be formed in a second polymer layer, preferably a
fluidic layer of the fluidic cartridge.
[0032] The bypass channel may be formed in the second polymer
layer. The valve membrane may comprise a thermoplastic elastomer.
The first polymer layer may comprise polypropylene. The second
polymer layer may comprise polypropylene. Polypropylene is an inert
material, which will not react with a fluid sample. It is also
readily bondable to other layers to facilitate manufacturing.
[0033] In a third aspect of the present invention there is provided
a method of metering a liquid sample in a fluidic cartridge
comprising a fluid pathway having a sample processing chamber
therein, an inlet valve upstream of the sample processing chamber
and an outlet valve downstream of the sample processing chamber, a
downstream sample processing region therein, and a bypass channel
coupled to the fluid pathway at a junction between the outlet valve
and the downstream sample processing region; the method comprising:
passing a liquid sample through the inlet valve, into the first
chamber, and through the outlet valve; closing the outlet valve and
evacuating surplus liquid sample downstream of the outlet valve
through the bypass channel to empty the fluid pathway downstream of
the outlet valve of fluid, thereby leaving a metered volume of
liquid sample in the fluid pathway between the inlet valve and the
downstream sample processing region; and opening the outlet valve
and delivering the metered volume of liquid sample to the
downstream sample processing region. This guarantees that
sufficient amount of fluid sample is delivered to the downstream
sample processing region for further processing. As will be
appreciated, the step of passing a liquid sample through the inlet
valve, into the first chamber, and through the outlet valve is
performed in a single step; that is, by applying a consistent fluid
pressure on the sample throughout the step (or a consistent average
pressure if bellows are used to move the sample).
[0034] The fluidic cartridge may further comprise at least one
compressible element downstream of the downstream sample processing
region; the step of passing a liquid sample through the inlet
valve, into the first chamber, and through the outlet valve may
further comprise compressing the compressible element as the liquid
sample passes downstream of the outlet valve; and the step of
evacuating surplus liquid sample downstream of the outlet valve may
further comprise the compressible element exerting a force against
the surplus liquid sample to expel it from the fluid pathway and
into the bypass channel. The compressible element utilises the
pressure created as fluid is advanced along the fluid pathway to
expel surplus fluid in the bypass channel, eliminating the need for
additional components in the fluidic cartridge for the purpose of
expelling surplus fluid.
[0035] The fluidic cartridge may further comprise a bypass valve in
the bypass channel, and wherein the method further comprises:
closing the bypass valve prior to the step of passing a liquid
sample through the inlet valve, into the first chamber, and through
the outlet valve; and wherein the step of evacuating surplus liquid
sample downstream of the outlet valve further comprises opening the
bypass valve. When the bypass valve is opened, in addition to
evacuating the bypass channels, the pressure in the compressible
elements and in the one or more fluid pathways equalises. When
there is a plurality of fluid pathways, this ensures that the fluid
advances equally along the plurality of fluid pathways.
[0036] The step of closing the inlet and outlet valves may comprise
closing the inlet and outlet valves simultaneously.
[0037] In a fourth aspect of the present invention there is
provided a method of expelling surplus liquid sample from a fluidic
cartridge comprising a fluid pathway having a downstream sample
processing region therein, an outlet valve upstream of the
downstream sample processing region, a bypass channel coupled to
the fluid pathway at a junction between the outlet valve and the
downstream sample processing region, and a compressible element
downstream of the downstream sample processing region; the method
comprising: passing a liquid sample through the outlet valve,
thereby compressing the compressible element as the liquid sample
passes downstream of the outlet valve; and closing the outlet valve
and evacuating surplus liquid sample downstream of the outlet valve
through the bypass channel by the compressible element exerting a
force against the surplus liquid sample to expel it from the fluid
pathway and into the bypass channel.
[0038] The fluidic cartridge further comprises a bypass valve in
the bypass channel, and wherein the method further comprises:
closing the bypass valve prior to the step of passing a liquid
sample through the outlet valve; and wherein the step of evacuating
surplus liquid sample downstream of the outlet valve further
comprises opening the bypass valve.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 is a schematic diagram of an exemplary fluidic
cartridge in which the invention may be provided.
[0040] FIG. 2 is a top view of an exemplary fluidic cartridge in
which the invention may be provided.
[0041] FIG. 3 is an exploded view of the exemplary fluidic
cartridge of FIG. 2.
[0042] FIG. 4 is a perspective view of the housing of the exemplary
fluidic cartridge of FIG. 2.
[0043] FIG. 5 is a perspective view of the blister sub-assembly of
the exemplary fluidic cartridge of FIG. 2.
[0044] FIG. 6A is a top view of the pneumatic layer of the
exemplary fluidic cartridge of FIG. 2.
[0045] FIG. 6B is a bottom view of the pneumatic layer of the
exemplary fluidic cartridge of FIG. 2.
[0046] FIG. 7 is a top view of the pneumatic foil of the exemplary
fluidic cartridge of FIG. 2.
[0047] FIG. 8A is a top view of the fluidic layer of the exemplary
fluidic cartridge of FIG. 2.
[0048] FIG. 8B is a bottom view of the fluidic layer of the
exemplary fluidic cartridge of FIG. 2.
[0049] FIG. 9 is a top view of the fluidic foil of the exemplary
fluidic cartridge of FIG. 2.
[0050] FIG. 10 is a top view of the electrode layer of the
exemplary fluidic cartridge of FIG. 2.
[0051] FIG. 11 is a section view of an advantageous valve
arrangement which may form an isolated inventive aspect.
[0052] FIG. 12 is a section view of another advantageous valve
arrangement which may form an isolated inventive aspect.
[0053] FIG. 13a is a section view of an advantageous inlet port
arrangement which may form an isolated inventive aspect.
[0054] FIG. 13b is a perspective section view of the inlet port
arrangement of FIG. 13a.
[0055] FIG. 14a is a section view of an advantageous capture column
arrangement which may form an isolated inventive aspect.
[0056] FIG. 14b is a perspective section view of a portion of the
capture column arrangement of FIG. 14a.
[0057] FIG. 15a is a section view of an advantageous waste chamber
arrangement which may form an isolated inventive aspect.
[0058] FIG. 15b is a perspective section view of the waste chamber
arrangement of FIG. 15a.
[0059] FIG. 16 is a schematic of a valve system according to a
first embodiment of the present invention.
[0060] FIG. 17a is a cross section of a valve suitable for the
valve system of FIG. 11 in a closed position.
[0061] FIG. 17b is a cross section of the valve of FIG. 17a in an
open position.
[0062] FIG. 18 is a schematic of the back end of the exemplary
cartridge including a second embodiment of a valve system of the
present invention.
[0063] FIG. 19 is a flow diagram of a method according to the
present invention.
[0064] FIG. 20 is a schematic of a valve system according to a
first embodiment of the present invention.
[0065] FIG. 21 is a section view of an exemplary valve.
[0066] FIG. 22 is a section view of another exemplary valve in an
open position.
[0067] FIG. 23 is a section view of the valve of FIG. 22 in an
intermediate position.
[0068] FIG. 24 is a section view of the valve of FIG. 22 in a
closed position.
[0069] FIG. 25 is a schematic diagram of an exemplary valve
system.
DETAILED DESCRIPTION
[0070] Embodiments of the invention will now be described in the
context of an exemplary fluid cartridge in which the invention is
implemented. Whilst not necessary to understand the present
invention, it is beneficial to provide general description of the
principles of the structure, manufacture, function and use of the
fluidic cartridge and associated methods for performing a test.
[0071] The exemplary fluidic cartridge and associated methods
chosen to illustrate the present invention are for the detection of
Chlamydia trachomatis bacterium using PCR amplification and
electrochemical detection. However, the skilled person would
understand that the invention is not limited to the exemplary
fluidic cartridge and associated methods, and is suitable for use
in with various different cartridges for a wide variety of sample
analysis techniques or biological assays; for example, assays of
target nucleic acid sequences in a liquid sample.
[0072] Those skilled in the art will understand that the devices
and methods of the invention described herein and illustrated in
the accompanying drawings are non-limiting exemplary embodiments
and that the scope of the present invention is defined solely by
the claims. The features illustrated or described in connection
with one exemplary embodiment may be combined with features of
other embodiments. Such modifications and variations are included
within the scope of the present disclosures.
[0073] The exemplary cartridge comprises: a fluidic portion through
which the sample flows and in which nucleic acid amplification and
detection take place; a pneumatic portion which controls flow
through the fluidic portion; and at least two electrodes which
provide a potential difference for the detection of an amplified
nucleic acid of interest. The fluidic portion and pneumatic portion
may be constructed of a fluidic layer, a fluidic foil, a pneumatic
layer and a pneumatic foil such as those described in relation to
the exemplary cartridge below. However, the fluidic portion does
not necessarily consist only of a fluidic layer and a fluidic foil
and the pneumatic portion does not necessarily consist only of a
pneumatic layer and a pneumatic foil. Rather, the layers may
interact to produce the fluidic portion and the pneumatic portion
such that parts of all or some of the layers make up each portion.
Rather than referring to the particular layers of the cartridge,
the fluidic portion refers to the particular areas of the cartridge
which provide the function of allowing controlled sample flow, and
the pneumatic portion refers to the particular areas of the
cartridge which provide the function of controlling the flow
through the fluidic portion.
[0074] The housing, fluidic portion and pneumatic portion are made
of plastic. By plastic is meant a synthetic or natural organic
material that may be shaped when soft and then hardened, including
resins, resinoids, polymers, cellulose derivatives, casein
materials, and protein plastics. Examples of plastics from which
the cartridge may be constructed include, but are not limited to
thermoplastics, for example polycarbonate, polyethylene
terephthalate, cyclic olefin copolymers such as Topaz,
acrylonitrile butadiene styrene, and thermoplastic elastomers, for
example polypropylene. Plastic housings, fluidic portions and
pneumatic portions can include components which are not made of
plastic (e.g. blisters made from metal foil, metallic inserts at
the sample inlet), but they are formed primarily from plastic. The
use of plastic materials facilitates economical manufacture of the
cartridges.
[0075] Whilst the pneumatic and fluidic foils may be made from a
metal foil, the preferred materials are plastic including those
mentioned above. In particular, it is preferred that foils are a
polyethylene terephthalate/polypropylene composite.
[0076] The target nucleic acid sequence is any nucleic acid to be
detected in a sample. The target nucleic acid(s) to be amplified
and detected in the cartridge will usually be DNA, but it is also
possible to amplify and detect RNA. In some embodiments a cartridge
may permit amplification and/or detection of both DNA and RNA
targets.
[0077] The liquid sample is the composition which is introduced
into the cartridge in order to determine whether the target nucleic
acid(s) of interest is/are present. The sample may be a composition
in which the nucleic acid to be detected is suspected to be present
(e.g. for clinical diagnosis), or may be a composition in which the
nucleic acid to be detected is potentially present (e.g. for
contamination testing).
[0078] The liquid sample can have various sources. For instance, it
can be material obtained from an animal or plant (e.g. for
diagnosis of infections or for genotyping). Such samples may be
obtained with minimal invasiveness or non-invasively, e.g., the
sample may be obtained from an animal using a swab, or may be a
bodily fluid. As an alternative, the sample may be material
obtained from food or water (e.g. for contamination testing). The
sample will usually include cells, and the target nucleic acid (if
present) can be extracted from these cells within the cartridge.
One skilled in the art will appreciate that samples can be diluted
or otherwise treated prior to being introduced into the cartridge,
but it is preferred that the cartridge can handle material which
has not been pre-treated in this way.
[0079] An animal from whom the sample is obtained may be a
vertebrate or non-vertebrate animal. Vertebrate animals may be
mammals. Examples of mammals include but are not limited to mouse,
rat, pig, dog, cat, rabbit, primates or the like. The animal may be
a primate, and is preferably a human. Thus the cartridge can be
used for clinical diagnosis of human samples.
[0080] In addition to analysing a sample, the cartridge can analyse
a positive and/or negative control to provide confirmation that the
cartridge is functioning as expected. The control(s) can be
introduced into the cartridge by a user, or can be included within
a cartridge before use.
[0081] The inclusion of an internal positive control nucleic acid
allows a user to identify whether a negative result for the sample
has been obtained because the nucleic acid amplification has been
unsuccessful (false negative). If the positive control nucleic acid
fails to be detected in the detection chamber, despite its presence
in an amplification chamber, the user will be able to identify the
test as a potential false negative result, and can perform another
test.
[0082] The inclusion of an internal negative control allows a user
to identify whether a positive result has been falsely obtained
because of the presence of contamination. A negative control can
involve performing PCR in a chamber in which no nucleic acid is
provided, or in which a sample undergoes an amplification reaction
without necessary components e.g. PCR without primers. If nucleic
acid is nevertheless detected in the detection chamber, despite its
intended absence in an amplification chamber, the user will be able
to identify the test as a potential false positive result, and can
perform another test.
[0083] A positive control nucleic acid may be any nucleic acid that
will not be found in a sample used in the cartridge. The internal
control DNA may be taken from a bacterium that is not pathogenic to
animals and which contains a nucleic acid that is highly specific
to the bacterium. One example of a possible bacterium from which
the control nucleic acid may be taken for an animal sample is
Pectobacterium atrosepticum, although any control nucleic acid may
be used that will not be present in a sample.
[0084] The fluidic portion of the cartridge comprises channels and
chambers through which sample flows. The flow of sample through the
cartridge is controlled in two ways. Firstly, the fluidic portion
has a gas inlet. The gas inlet is connected to a gas supply, and
injection of gas into the fluidic portion via this inlet allows the
sample to be pushed downstream through the cartridge, towards the
detection chamber. The gas supply may be provided by the reader. As
an alternative, the gas supply may be an on-board gas supply.
Preferably, the gas supply is provided by an external source and
the gas inlet is connected to a pneumatic circuit such that the gas
supply is provided via a pneumatic inlet on the cartridge.
Secondly, at least one pneumatically controlled valve controls
local movement of the sample through the fluidic portion. The
pneumatically controlled valve(s) may be controlled independently
of other pneumatically controlled valves and may be controlled
independently of the gas supply that generally causes downstream
movement of the sample via the gas inlet. The gas inlet and the
pneumatically controlled valve(s) also permit sample to be flushed
through the fluidic portion e.g. to exclude excess volumes of
material. The fluidic portion also has an exhaust which allows air
and waste material to exit the channels and chambers of the fluidic
portion without a build-up of pressure occurring in the cartridge.
Preferably, the exhaust comprises a waste chamber and/or a waste
vent.
[0085] The fluidic portion of the cartridge includes reagents
and/or physical components for cell lysis and nucleic acid
separation. These may be any reagents or physical components that
are capable of lysing cells and separating nucleic acids from cell
debris and other cellular components. For instance, they may
comprise (i) a lysis buffer which is capable of causing lysis of
target cells which may be present in the sample e.g. buffers
including a detergent such as nonyl phenoxypolyethoxylethanol
(available as NP-40) or t-octylphenoxypolyethoxyethanol, (available
as Triton X 100), or including guanidine thiocyanate, and/or (ii) a
capture support or column which specifically binds nucleic acids
but does not bind other undesired cellular components (e.g.
proteins and lipids). The capture column comprises a capture filter
and may additionally comprise a depth filter. The filters may be
made of glass fibres (available as Whatman filters), or may be made
of silica, although any column or support which is capable of
separating nucleic acids from other cellular components may be
used. Elution using a wash buffer to remove cell debris and other
cellular components, followed by elution using an elution buffer to
elute the separated nucleic acids from the capture support or
column can be undertaken such that the capture column can separate
nucleic acids from cell debris and other cellular components.
[0086] A channel through which the sample flows fluidly connects
the sample inlet to at least one amplification chamber where
nucleic acid amplification can take place. The purpose of the
amplification chamber(s) is to permit amplification of any target
nucleic acid of interest that is present in the sample (and, where
present, any positive control nucleic acid). Any nucleic acid
amplification method may be used and these are described in more
detail below in relation to an exemplary cartridge. The different
nucleic acid amplification reagents that are required for different
nucleic acid amplification methods are well known in the art. These
reagents are provided in or upstream of the amplification
chamber(s) such that the sample (and any positive control) includes
all necessary reagents for nucleic acid amplification once it
reaches the amplification chamber. Adaptation of a nucleic acid
amplification method according to the target nucleic acid to be
detected is also well known in the art (e.g. design of primers).
The skilled person would therefore be able to adapt the reagents
for nucleic acid amplification accordingly. The term "chamber" does
not denote any particular size or geometry, but instead it means a
region within the fluidic portion which is designed to permit
nucleic acid amplification to occur. Thus, for instance, it could
be a region in which the sample can be fluidically isolated (e.g.
via the use of pneumatically controlled valves) while the steps
required for nucleic acid amplification (e.g. thermocycling, etc.)
occur, and it can be located within the cartridge so that it is in
the proximity of any external resources that are needed (e.g. next
to a heat source within a cartridge reader, thereby permitting
thermal cycling to occur).
[0087] Multiple test amplification channels and/or chambers may be
included in the cartridge. The different test amplification
channels and/or chambers may include reagents required to amplify
different nucleic acids of interest. Therefore using multiple
amplification test channels and/or chambers allows multiple tests
to be performed on a single cartridge, simultaneously (including
any controls). As an alternative, reagents for amplification of
multiple different nucleic acids may be present in a single
amplification chamber, and the different nucleic acids (whether
multiple target nucleic acids, or a target nucleic acid and a
control nucleic acid) may be amplified simultaneously in the same
amplification chamber.
[0088] A further channel through which the sample flows after
nucleic acid amplification fluidly connects the at least one
amplification chamber to at least one detection chamber where the
results of nucleic acid amplification can be detected. In or
upstream of the detection chamber are reagents for nucleic acid
detection such that the sample includes all necessary reagents for
the detection once it reaches the detection chamber. The reagents
for nucleic acid detection may be specific for the particular
target nucleic acid, i.e. they may allow for detection of the
presence of the specific nucleic acid sequence. As an alternative,
the reagents for nucleic acid detection may be generic reagents to
detect the presence of any nucleic acids. Such generic reagents may
be used if all nucleic acids other than the target nucleic acid are
removed prior to detection. For example, this may be achieved by
providing a nuclease that is capable of hydrolysing all nucleic
acids present in the sample other than the target nucleic. The
amplified target nucleic acid can be protected from hydrolysis, for
example by inclusion of chemical modifications in the primers which
are incorporated into the amplified product and which cannot be
hydrolysed. Reagents for nucleic acid detection are described below
in relation to an exemplary cartridge but usually comprise a probe
including a label. The probe is capable of hybridising to the
amplified nucleic acid which has been amplified in the
amplification chamber(s). Following hybridisation of the probe to
the amplified nucleic acid, the detection of the nucleic acid may
occur via a detectable change in the signal from the label. In some
embodiments the change may be caused by hydrolysis of the probe.
Where the probe is hydrolysed, hydrolysis is usually achieved using
a double strand specific nuclease, which can be an exonuclease or
an endonuclease. Preferably, the nuclease is T7 endonuclease. The
signal from the label is capable of undergoing a change following
hydrolysis of the probe. This is due to a change in the environment
of the label when it moves from being bound to the rest of the
probe to being free from the rest of the probe or bound to a single
nucleotide or a short part of the probe. Further details of the
types of probes and detection methods that may be used can be found
in Hillier et al. Bioelectrochemistry, 63 (2004), 307-310. As an
alternative, methods for causing a detectable change in the signal
from the label which do not rely on hydrolysis of the probe may be
used e.g. see Ihara et al. Nucleic Acids Research, 1996, Vol. 24,
No. 21 4273-4280. This change in environment of the label leads to
a change in the signal from the label. The change in signal from
the label can be detected in order to detect the presence of the
nucleic acid of interest.
[0089] Where a positive control nucleic acid is used, the reagents
for nucleic acid detection will additionally include a positive
control probe including a label. The positive control probe is
capable of hybridising to the amplified control nucleic acid. The
signal provided by the labels of the positive control and target
probes may be the same, but present in separate detection chambers
such that the signals corresponding to the control and test nucleic
acids can be distinguished. As an alternative, the signal provided
by the labels of the control and target probes may be different,
such that the signals are distinguishable from one another, even if
the probes are present in the same detection chamber.
[0090] Multiple test detection channels and/or chambers may be
included in the cartridge. The different test detection channels
and/or chambers may include reagents required to detect different
nucleic acids of interest. Therefore using multiple detection test
channels and/or chambers allows multiple tests to be performed on a
single cartridge, simultaneously. As an alternative, reagents for
detection of multiple different nucleic acids may be present in a
single detection chamber, and the different nucleic acids (whether
multiple target nucleic acids or a target nucleic acid and a
control nucleic acid) may be detected simultaneously in the same
detection chamber.
[0091] The label is detectable by use of the cartridge's
electrodes, and so the label will usually be an electrochemical
label, such as a ferrocene. Examples of labels which may be used
can be found in WO03/074731, WO2012/085591 and PCT/GB2013/051643.
Signal emitted by the label can be detected by a cartridge
reader.
[0092] The pneumatic portion of the cartridge comprises at least
one pneumatic circuit which each control at least one pneumatically
controlled valve. The pneumatic portion controls sample flow
through the cartridge by the opening and closing of pneumatically
controlled valves. The opening and closing of the valves is
controlled by changes in pneumatic pressure in the pneumatic
circuit that is applied through a pneumatic pressure inlet.
Usually, the cartridge contains many pneumatically controlled
valves. The pneumatically controlled valves may be controlled by
separate pneumatic pressure inlets. These valves can be used to
prevent downstream movement of sample through the fluidic portion
until necessary steps have been performed and/or to prevent
unwanted reverse movement of sample upstream. For example, a valve
may be provided upstream of the at least one amplification chamber
in order to prevent downstream movement into the at least one
amplification chamber until cell lysis and nucleic acid separation
has taken place. Following cell lysis and nucleic acid separation
the valve upstream of the at least one amplification chamber may be
opened in order to allow downstream flow. It can then be closed
again, to prevent backflow out of the chamber back towards the
sample inlet.
[0093] The cartridge comprises at least two electrodes which can
provide a potential difference across the at least one detection
chamber. The potential difference causes current to flow through
the at least one detection chamber, thereby permitting the
detection of signal from electrochemically active labels.
[0094] An exemplary cartridge which operates according to the above
description will now be described with reference to the
accompanying drawings.
[0095] 1. The Exemplary Cartridge
[0096] 1.1 Overview
[0097] The exemplary cartridge described below is intended to be a
single-use, disposable cartridge for performing a test on a sample
introduced into the cartridge. The exemplary cartridge is a fluidic
cartridge with channels of an appropriate scale (as detailed
hereafter). However, the invention may be performed on a
microfluidic device, or an LOC. Once the test has been run, it is
preferred that the cartridge is disposed of. However, if desired,
the cartridge may be sent for re-processing to enable it to be used
again.
[0098] It is preferred that the cartridge comprises all of the
biological agents necessary for conducting the test of choice. For
example, the exemplary cartridge is used for detecting the
presence, absence or amount of a pathogen of interest. Any pathogen
may be detected. Examples of pathogens which may be detected by the
cartridge are Chlamydia trachomatis, Trichomonas vaginalis,
Neisseria gonorrhoea, Mycoplasma genitalium and methicillin
resistant Staphylococcus aureus. To that end the cartridge
comprises reagents for nucleic acid amplification. Nucleic acid
amplification may be performed using any nucleic acid amplification
method. The nucleic acid amplification method may be a
thermocycling method in which the temperature at which the method
is performed is varied such that different steps of the
amplification are able to take place at different temperatures
within the cycle. For example melting, annealing of primers and
extension may each be performed at different temperatures. By
cycling through the temperatures, the timing of each of the steps
of the method can be controlled. As an alternative, the nucleic
acid amplification may be an isothermal method in which the
temperature is kept constant. In both the thermocycling and the
isothermal nucleic acid amplification methods, the temperature is
controlled during nucleic acid amplification.
[0099] Examples of nucleic acid amplification methods are the
polymerase chain reaction (PCR), the ligase chain reaction (LCR),
strand displacement amplification (SDA), transcription mediated
amplification, nucleic acid sequence-based amplification (NASBA),
helicase-dependent amplification and loop-mediated isothermal
amplification. The reagents for nucleic acid amplification will
vary depending of the nucleic acid amplification method used but
include a polymerase and nucleotide triphosphates.
[0100] As explained below, the cartridge also comprises detection
reagents which are capable of detecting the presence or absence of
amplified nucleic acids which are the product of the nucleic acid
amplification method. The reagents for nucleic acid detection
comprise a probe which is capable of hybridising to the amplified
nucleic acid. The probe includes a ferrocene label.
[0101] Following hybridisation of the probe to the amplified
nucleic acid, the detection of the nucleic acid occurs via a
detectable change in the signal from the label. The change is
caused by hydrolysis of the probe, which is achieved using a double
strand specific nuclease. The nuclease is a T7 endonuclease. The
ferrocene gives different electrochemical signals when it is part
of a probe or when it is attached only to a single nucleotide, and
so hydrolysis is easily detected. Thus, the change in signal from
the label permits detection of the presence of the nucleic acid of
interest.
[0102] The electrodes allow the detectable change in the signal
from the label, which occurs in the presence of the target nucleic
acid, to be detected.
[0103] The cartridge is configured for use with a cartridge reader
(not shown). The cartridge comprises a number of pneumatic,
mechanical, thermal and electrical interfaces (described in more
detail below) through which the reader interacts with the cartridge
to perform the test. Hence, in use, the cartridge would be inserted
into the reader, and the reader would be activated to begin
interacting with the cartridge via the interfaces to perform the
test. For the purposes of understanding the present invention, it
is not necessary to describe exactly how the cartridge interacts
with the reader to conduct a particular test and provide the test
results, but an overview of an exemplary operation of a cartridge
is provided hereafter.
[0104] 1.2 Schematic Diagram of the Exemplary Cartridge
[0105] Before explaining the structure and arrangement of the
components of an exemplary fluid cartridge in detail, it is helpful
to describe the layout of the exemplary cartridge at a high level
with reference to the schematic shown in FIG. 1.
[0106] It is convenient to consider the overall layout of the
cartridge in terms of the flow of liquids, including the liquid
sample, through the cartridge. Unless otherwise specified
hereafter, the passage of liquids including the liquid sample and
the liquid buffers is referred to as the `fluid pathway` which has
an upstream end and a downstream end. Unless otherwise specified
hereafter, `downstream` generally refers to the direction of flow
of the liquids and `upstream` refers to the direction opposite the
direction of flow. The fluid pathway in the exemplary cartridge may
have different branches (and thus form different fluid pathways),
but all pathways have a recognisable direction of flow which permit
a skilled person to identify the upstream and downstream
directions. However, there is an exception to this general
definition, which is when the liquid sample is pumped between the
mixing chamber 10 and the bellows 20. In this case, fluid is
intermittently pumped back upstream in the opposite direction to
its general direction of fluid flow, which is downstream. This
mixing serves to mix the lysis and sample and to rehydrate the
internal control.
[0107] The liquid sample is introduced into the cartridge at a
sample mixing chamber 10 through an entry port. A particular
arrangement of a preferred entry port may itself form an isolated
inventive aspect of the cartridge, as described further in section
3, below. A sample indicator 12 is fluidly coupled to the sample
mixing chamber 10 such that a sample introduced into the sample
mixing chamber 10 is visible in the sample indicator 12. Also
connected to the sample mixing chamber 10 is a blister 14
containing a lysis buffer. The lysis buffer comprises guanidine
thiocyanate. Once the sample has been introduced into the sample
mixing chamber 10, and a test is started, the lysis blister 14 is
collapsed so as to expel the lysis buffer into the sample mixing
chamber 10 where it mixes with the liquid sample introduced
therein.
[0108] Downstream of the sample mixing chamber 10, along a main
channel 16, is a coarse filter 18. The coarse filter 18 filters out
any large debris in the liquid sample, such as skin or bodily hair,
as the liquid sample passes through main channel 16.
[0109] Downstream of the coarse filter 18, along the main channel
16, is a bellows 20 having an upstream bellows valve 22a and a
downstream bellows valve 22b. As described in more detail below,
the bellows 20, together with its upstream and downstream valves
22a-b, is capable of pumping the liquid sample from the upstream
end of the fluid pathway (i.e. from the sample mixing chamber 10)
to the downstream end. In summary, this is achieved by virtue of
flexible membranes within the bellows 20 and the upstream and
downstream bellows valves 22a-b which actuate to create local
pressure differentials to, on the one hand, draw in the liquid
sample from the sample mixing chamber 10 into the bellows 20 and,
on the other hand, from the bellows 20 further downstream through
the main channel 16. This is achieved by carefully choreographed
pneumatic actuation of the flexible membranes in the valves.
Particular arrangements of a preferred valve may themselves form
isolated inventive aspects of the cartridge, as described further
in section 3, below.
[0110] Downstream of the bellows along the main channel 16 is a
capture column 24. The purpose of the capture column 24 is to
separate nucleic acids from cell debris and other cellular
components. The capture column comprises a capture filter and a
depth filter both made of glass fibres. A particular arrangement of
a preferred capture column may itself form an isolated inventive
aspect of the cartridge, as described further in section 3,
below.
[0111] Two branch channels 26, 28 join the main channel 16 between
the downstream bellows valve 22b and the capture column 24. The
purpose of the branch channels is to introduce liquid buffers
necessary for performing the desired test. For example, with the
test conducted by the exemplary cartridge, it is necessary to
introduce an elution buffer and a wash buffer into the main channel
once the sample has passed through. The wash buffer is contained in
a wash buffer blister 30 and the elution buffer is contained in an
elution buffer blister 32. The introduction of the wash buffer and
elution buffer into the main channel 16 is controlled by wash
buffer valve 34 and elution buffer valve 36, respectively. At the
appropriate point in the test, the wash and elution buffer blisters
30, 32 are collapsed so as to expel the wash and elution buffers
into the branch channels 26, 28 and thence into the main channel 16
through the wash and elution buffer valves 34, 36.
[0112] Downstream of the capture column 24, along a waste branch
16a of the main channel 16, is a waste chamber 38. A particular
arrangement of a preferred waste chamber may itself form an
isolated inventive aspect of the cartridge, as described further in
section 3, below. The purpose of the waste chamber 38 is to collect
the cell debris and cellular components other than nucleic acids
and contain them, thereby preventing them from entering the test
channel 54a or the control channel 54b. The waste chamber 38 is
vented to atmosphere through a waste vent 40, and an aerosol
impactor 42 is provided between the waste chamber 38 and the waste
vent 40 to prevent particulate matter from escaping from the waste
chamber 38 into the atmosphere. A waste chamber valve 44 in the
main channel waste branch 16a of the main channel 16 permits and
prevents fluids passing into the waste chamber 38 at appropriate
points during the test.
[0113] Downstream of the capture column 24, along an elution branch
16b of the main channel 16, is an elution chamber 46. The purpose
of the elution chamber 46 is to allow the sample preparation to
settle and for bubbles to disperse before the sample enters the
amplification chambers. An elution chamber valve 48 in the elution
branch 16b of the main channel 16 permits and prevents fluids
passing into the elution chamber 46 at appropriate points during
the test.
[0114] Downstream of the elution chamber 46 is a convoluted mixing
channel 52. Here the prepared sample is mixed prior to passing
through the isolation valve 50.
[0115] In the present application, the components upstream of the
isolation valve 50 are referred to as being comprised in the `front
end` of the cartridge, whilst the components downstream of the
isolation valve 50 are referred to as being comprised in the `back
end` of the cartridge. Broadly speaking, the liquid sample is
prepared for analysing in the front end of the cartridge, and the
analysis is carried out on the sample in the back end of the
cartridge.
[0116] The isolation valve 50 is open to permit the prepared liquid
sample to pass from the front end to the back end of the cartridge.
At an appropriate point in the test, after the liquid sample has
been prepared and is within the back end of the cartridge for
analysis, the isolation valve 50 is closed to prevent any of the
sample from re-entering the front end. Once the isolation valve 50
is closed, it cannot be opened again. The isolation valve 50 also
acts as a safeguard in case of a power failure, wherein the reader
closes the isolation valve 50 to prevent leakage.
[0117] Downstream of the isolation valve 50, the fluid pathway
splits into an amplification test channel 54a and an amplification
control channel 54b. Each of the amplification channels 54a-b
comprises an amplification chamber 56a-b having an amplification
chamber inlet valve 58a-b and an amplification chamber outlet valve
60a-b. Any nucleic acid amplification method may be performed in
the nucleic acid amplification chamber. If PCR is used, the nucleic
acid amplification chambers contain a thermostable DNA polymerase,
dNTPs, a pair of primers which are capable of hybridising to the
nucleic acid to be amplified. Optionally, the nucleic acid
amplification chambers may additionally contain buffer salts,
MgCl.sub.2, passivation agents, uracil N-glycosylase and dUTP. An
example of a thermostable DNA polymerase that may be used is Taq
polymerase from Thermus aquaticus.
[0118] Each of the nucleic acid amplification chambers in the
exemplary cartridge comprises reagent containment features in the
form of first and second shallow wells formed in the fluidic layer.
The reagents to be used in the cartridge are spotted in the wells.
In the exemplary cartridge, the test-specific reagents and the
generic reagents are isolated from each other by spotting each in a
different well. Hence, the test-specific reagents are spotted in a
first well in the chamber and the generic reagents are spotted in a
second well in the chamber. By spotting the reagents separately, it
is easier to swap the test-specific reagents during manufacture for
a different set of test-specific reagents, so as to perform a
different test, whilst keeping the generic reagents as they
are.
[0119] In the exemplary cartridge, the ratio of nucleic acid
amplification chambers to detection chambers is 1:2. The prepared
sample enters the back end of the cartridge at the isolation valve
50 and is split into two nucleic acid amplification chambers. After
processing, the each of the two processed measures of sample from
the nucleic acid amplification chamber is split into two detection
chambers. Therefore, for each sample introduced into the exemplary
cartridge, four detection chambers may be filled from two nucleic
acid amplification chambers, thus facilitating duplex amplification
and 4-plex detection.
[0120] However, it will be appreciated that one or three or more
nucleic acid amplification chambers may be provided to provide any
level of multiplexing desired, and that the number of the detection
chambers provided may be adjusted accordingly to maintain a 1:2
ratio of nucleic acid amplification chambers to detection
chambers.
[0121] The ratio 1:2 is preferred for the exemplary cartridge
because such a ratio allows twice the number of target nucleic
acids to be assayed compared to the number of different labels
required for detection in the detection chambers. However, it will
be appreciated that the ratio may be changed depending on the
number of labels and PCR targets for the liquid sample. For
instance, the ratio may be 1:1, 1:3 or 1:n such that there are n
detection chambers branching from the main channel of each fluid
pathway when there are n times as many multiplexed PCR targets for
the number of labels.
[0122] PCR primers specific for Chlamydia trachomatis are dried
down in the amplification chamber in the amplification test channel
together with the other reagents required for nucleic acid
amplification. PCR primers specific for a positive control nucleic
acid are dried down in the amplification chamber in the
amplification control channel together with the other reagents
required for nucleic acid amplification. A positive control nucleic
acid is also provided in the amplification chamber in the
amplification control channel, taken from Pectobacterium
atrosepticum. The dried down reagents are reconstituted when the
liquid sample reaches them.
[0123] Downstream of the amplification chamber outlet valves 60a-b
each of the amplification channels 54a-b splits into two further
detection channels, leading to two detection chambers for each
amplification chamber, giving a total of four detection chambers
62a-d in total. The reagents for nucleic acid detection, including
the target probe, are dried down in the detection chambers 62a-d
downstream of the test amplification chamber 56a or 56b. The
reagents for nucleic acid detection including the control probe are
dried down in the detection chambers downstream of the control
amplification chamber 56a or 56b (whichever is not the test chamber
mentioned above). Each detection chamber 62a-d is provided with its
own gas spring 64a-d which forms a dead end at the downstream end
of the fluid pathway.
[0124] Reagents for nucleic acid detection are provided in
detection chambers. The reagents for nucleic acid detection include
probes having a ferrocene label. These probes are capable of
hybridising to the amplified nucleic acids. Following hybridisation
of the probes to the amplified nucleic acids, the probes are
hydrolysed by a double strand specific nuclease which causes the
label to be freed from the rest of the probe. As explained above,
freeing of the label from the rest of the probe causes a detectable
change in the signal from the label. The control probe is provided
in separate detection chambers to the target probe and detection of
the target nucleic acid and the control nucleic acid take place in
different detection chambers, such that the signals are
distinguishable from one another.
[0125] Downstream of the amplification outlet valves 60a-b, but
upstream of the forks creating the four detection channels, two
bypass channels 66a-b respectively join the two amplification
channels 54a-b. The purpose of the bypass channels 66a-b is to
remove excess liquid sample within the amplification channels 54a-b
before the liquid sample enters the detection chambers 62a-d. The
bypass channels 66a-b connect to a bypass valve 68, which is also
fluidly coupled to the elution chamber branch 16b of the main
channel 16, downstream of the isolation valve 50, before the
channel splits into amplification channels 54a and 54b.
[0126] A particular arrangement of a preferred chamber in the
cartridge, such as the first and second amplification chambers or
the first to fourth detection chambers, may itself form an isolated
inventive aspect of the cartridge, as described further in section
3, below.
[0127] It will be appreciated that the number of amplification
chambers, and the number of detection chambers in the exemplary
cartridge may vary depending on the preferred implementation.
Moreover, other configurations of channels, chambers, valves and so
on are possible without departing from the scope of the invention,
as defined by the claims.
[0128] The physical structure and operation of the various
components of the exemplary cartridge introduced above will now be
explained with reference to FIGS. 2 to 10.
[0129] 1.3 Physical Structure of an Exemplary Cartridge
[0130] 1.3.1 Overview and External Features of the Exemplary
Cartridge
[0131] An exemplary cartridge is shown in FIG. 2. As described
above, the reader interacts with the cartridge through a plurality
of interfaces. The interfaces shown in the exemplary cartridge 100
are: a pneumatic interface 101; an electrical interface 102; a
bypass valve interface 103; and an isolation valve interface 104.
Each of these interfaces is described in more detail below. It will
be appreciated that more or fewer interfaces could be provided,
depending on the preferred implementation.
[0132] Also provided in the cartridge, but not shown, is a thermal
interface. The thermal interface allows the temperature of the
amplification chambers to be regulated to allow nucleic acid
amplification to take place.
[0133] The exemplary cartridge 100 shown in FIG. 2 comprises an
insertion end 105 for insertion into the reader, and a
non-insertion end 106. Proximate the non-insertion end 106 is a
sample inlet 107 for introducing a sample into the sample mixing
chamber 10. In the exemplary cartridge, the sample will usually
include cells, and the target nucleic acid (if present) can be
extracted from these cells, but other fluid samples such as swab
eluate, urine, semen, blood, saliva, stool sweat and tears could be
used in other implementations. The sample may be introduced into
the sample mixing chamber 10 through the sample inlet 107 using a
pipette, for example.
[0134] The exemplary cartridge 100 and reader are configured such
that when the cartridge is inserted into the reader, all of the
aforementioned interfaces are actuatable by the reader. On the
other hand, the sample inlet 107 remains external to the reader
such that a sample may be introduced into the sample mixing chamber
10 whilst the cartridge is inserted into the reader.
[0135] The exemplary cartridge 100 shown in FIG. 2 further
comprises a sample indicator window 109, through which the sample
indicator 12 is visible to determine whether a sample has been
introduced into the sample mixing chamber 10.
[0136] All of the pneumatic, mechanical and electrical interfaces
in the exemplary cartridge 100 are located on the same face of the
cartridge, in this case the top face 110. The thermal interface
(not shown) is provided on the bottom face of the cartridge. This
simplifies the design of the reader, which may this provide the
associated pneumatic, mechanical and electrical parts which
interact with those interfaces in the same region of the reader,
thereby making best use of space. It also enables the thermal part
of the reader to be provided away from the pneumatic, mechanical
and electrical parts.
[0137] 1.3.2 Internal Components of Cartridge
[0138] The exemplary cartridge 100 shown in FIG. 2 is formed from
various components which shall now be described. FIG. 3 shows an
exploded view of the exemplary cartridge 100 of FIG. 2. The
cartridge 100 comprises, from top to bottom, a housing 111, a
blister sub-assembly 112, a pneumatic foil 113, a pneumatic layer
114, a fluid layer 115 and a fluidic foil 116. Also shown in FIG. 3
is an electrode layer 117, two filters 118 and a plurality of
absorbent pads 119, which will be described in more detail
below.
[0139] The housing 111 is manufactured from acrylonitrile butadiene
styrene. The pneumatic and fluidic foils 113, 116 are manufactured
from a polyethylene terephthalate/polypropylene composite. The
pneumatic and fluidic layers 114, 115 are manufacture from
polypropylene.
[0140] With the exception of the housing 111, filters 118 and pads
119, each of the components mentioned in the previous paragraph is
adhered to its adjacent component or components. Hence, the blister
sub-assembly 112 is adhered to the pneumatic foil 113, which is
adhered to the pneumatic layer 114, which is adhered to the fluidic
layer 115, which is adhered to the fluidic foil 116. The electrode
layer 117 is adhered to fluidic layer 115 also.
[0141] The adhesion of the layers to each other provides a series
of fluid-tight channels in the cartridge, together with associated
chambers, valves, pumps, bellows and other components. The channels
passing a liquid sample therethrough are liquid-tight and the
channels passing a gas therethrough are gas-tight. Optionally, all
components are both liquid tight and gas-tight. For example,
recesses and openings formed in one or both sides of the pneumatic
and fluidic layers create, when sandwiched together and adhered to
the pneumatic and fluidic foils, respectively, the shapes necessary
to provide the aforesaid channels, chambers, valves, pumps, bellows
and other components.
[0142] Each of the components referred to above in FIG. 3 will now
be described in more detail.
[0143] 1.3.3 Housing 111
[0144] FIG. 4 shows housing 111 in more detail. As shown, housing
111 comprises a generally rectangular upper surface 120 and walls
121 depending therefrom on all four sides (two of which are visible
in FIG. 4). A principal purpose of the housing 111 is to protect
certain components of the cartridge, most notably the blister
sub-assembly 112 and the isolation valve interface 104. It will
therefore be noted that the housing 111 is shorter than the
pneumatic and fluidic layers 114, 115 such that it overlies only a
portion of those layers when the cartridge 100 is assembled. In the
exemplary cartridge 100, the pneumatic interface 101, electronic
interface 102, and bypass valve interface 103 are not covered by
the housing 111 to provide ease of access by the reader.
[0145] The upper surface 120 of the housing 111 has three apertures
122a-c therein, each having walls depending from the peripheries of
the apertures to form, when the cartridge is assembled, three
recesses. The purpose of the recesses is to house the blisters of
the blister sub-assembly 112 such that the blisters may be accessed
and pressed by the reader, but are otherwise protected from
accidental impact. Naturally, since the exemplary cartridge
comprises three blisters, the housing 111 comprises three
corresponding apertures 122a-c forming three corresponding
recesses. It will be appreciated that more or fewer blisters,
apertures and recesses may be provided, depending on the preferred
implementation. Alternatively, the housing 111 could comprise a
single aperture forming a single recess housing all available
blisters.
[0146] The side walls 121 of the housing 111 which run along the
length of the housing 111 between the insertion end 105 and the
non-insertion end 106 of the cartridge 100 comprise flanges 123
along at least a portion of their lower edges. The purpose of the
flanges 123 is two-fold. Firstly, they comprise one or more windows
124a-b for receiving a corresponding number of tabs formed in the
pneumatic layer 114 to hold the cartridge 100 together. Secondly,
the flanges 123 are dimensioned so as to protrude beyond the lower
surface of the fluidic foil 116 when the cartridge is assembled,
such that the fluidic foil 116 is suspended above a flat surface on
which the cartridge 100 is placed. This prevents accidental damage
to the fluidic foil 116 which could otherwise result.
[0147] Although in the exemplary cartridge depicted in FIG. 4
flanges 123 are provided along substantially the length of two
opposing sides of the cartridge, it will be appreciated that
flanges may be provided along three or four edges of the cartridge
and still suspend the foil above a flat surface on which the
cartridge is placed. Similarly, although the cartridge depicted in
FIG. 4 shows flanges 123 extending along substantially the entire
length of the edge, a flange which extends only partially along an
edge may be provided, or multiple flanges may be provided along
each edge.
[0148] The housing 111 further comprises, at the non-insertion end
106, a grip 125 to facilitate insertion of the cartridge into and
removal of the cartridge 100 from the reader by hand. The grip 125
comprises a series of ridges and grooves formed in the housing 111,
but alternative structures to increase friction, such as knurls,
are also possible.
[0149] The housing 111 further comprises a sample inlet aperture
126 through which a sample may be introduced into the sample mixing
chamber 10 of the cartridge 100 using a pipette, for example.
Surrounding the inlet aperture 126 for a given diameter is a basin
127 recessed into the upper surface 120 of the housing 111 to
accommodate a certain amount of spillage of the liquid sample.
Whilst the basin 127 of the exemplary embodiment is substantially
flat, it may be sloped toward the inlet aperture 126, such that any
spillage drains through the inlet aperture 126.
[0150] The exemplary housing 111 further comprises a plurality of
cut-outs: a first cut-out 128 forming the sample window 109, and a
second cut-out 129 to provide access to the isolation valve
interface 104. As with the recesses which protect the blisters, by
providing access to the isolation valve interface 104 only through
a cut-out 129 in the housing 111, the isolation valve interface 104
is protected to some extent from accidental impact, which could
actuate the isolation valve and render the cartridge
inoperable.
[0151] 1.3.4 Blister Sub-Assembly 112
[0152] FIG. 5 shows the blister sub-assembly 112 in more detail.
The blister sub-assembly 112 may be manufactured separately, during
which the blisters are pre-filled with the liquid reagents
necessary for conducting the preferred test, and subsequently
adhered to the pneumatic foil 113.
[0153] Blister sub-assemblies (or `blister packs`) are familiar to
a skilled person. A blister is a collapsible chamber for containing
a liquid, which may be expelled from the blister by pressing on the
blister and thereby collapsing it. In typical blister packs, the
chamber of a blister is sealed by a foil or other frangible layer
which ruptures once the pressure inside the chamber reaches a
particular magnitude as the blister is collapsed.
[0154] In the exemplary cartridge, the blister sub-assembly 112
comprises three blisters 130a-c. These contain, respectively, the
lysis buffer which comprises reagents capable of performing cell
lysis, the wash buffer and the elution buffer.
[0155] The exemplary blister sub-assembly 112 comprises a substrate
131 onto which the aforementioned blisters 130a-c are formed by a
deformable polymeric layer which is shaped to provide the chambers.
Three apertures 132a-c, corresponding to the three blisters 130a-c,
pass through the substrate 132. Each of the apertures is covered by
the deformable polymeric layer forming the chamber, which thereby
connects the aperture to the chamber but for a seal 133a-c between
the respective apertures 132a-c and chambers. Upon application of a
suitable pressure on the blister 130a-c, the seal 133a-c breaks,
thereby causing the liquid contents of the blister to be ejected
from the blister and to flow through the aperture 132a-c in the
substrate 131 out of the blister sub-assembly.
[0156] As shown, the seals 133a-c at least partially surround the
periphery of the chambers, where they meet the substrate 131. At
the point in each seal 133a-c which is designed to break (thereby
forming the liquid passageway between the aperture 132a-c and
chamber), the seal 133a-c may be weaker than the rest of the
periphery. This ensures that the correct part of the seal 133a-c
breaks when the suitable pressure is applied.
[0157] The blisters may be collapsed by the reader when the
cartridge is inserted therein. One or more mechanical actuators
(such as a foot) may be applied by the reader into the recess so as
to collapse the blister.
[0158] The blister sub-assembly 112 further comprises two reference
holes 134a-b configured to permit an assembly fixture to provide a
reference to facilitate positioning of the assembly during
manufacture.
[0159] 1.3.5 Pneumatic Layer 114
[0160] FIGS. 6A and 6B show the pneumatic layer 114 in more detail.
FIG. 6A is a top view of the pneumatic layer and FIG. 6B is a
bottom view. The pneumatic layer 114 is comprised of a rigid
plastic layer 135 which, in certain places, is overmoulded with a
plurality of flexible membranes to form certain components when the
cartridge is assembled. The flexible membranes are manufactured
from a thermoplastic elastomer.
[0161] The rigid plastic layer 135 has a plurality of
differently-shaped recesses therein and apertures therethrough. In
combination with the fluidic layer 115, certain recesses within,
and/or apertures through, the rigid plastic layer 135 form a number
of components, including: the sample mixing chamber 136; the waste
chamber 137; the capture column 138; the elution chamber 139; the
first and second amplification chambers 140a-b; and the first to
fourth detection chambers 141a-d. An aperture 142 is also provided
to give access to the electrode layer 117.
[0162] In combination with the overmoulded flexible membranes and
the pneumatic foil 113, certain other apertures through the rigid
plastic layer form a number of other components, including: the
upstream bellows valve 142; the bellows 143; a pneumatic interface
144; the downstream bellows valve 145; the wash buffer inlet valve
146; the wash buffer air inlet valve 146a; the elution buffer inlet
valve 147; the elution buffer air inlet valve 147a; the waste
chamber valve 148; the elution chamber valve 149; the isolation
valve 150; the first and second amplification chamber inlet valves
151a-b; and first and second amplification chamber outlet valves
152a-b. A further aperture, in combination with an overmoulded
flexible membrane (but not the pneumatic foil) forms a bypass valve
153.
[0163] With the exception of the isolation valve 150 and the bypass
valve 153, the valves formed in the pneumatic layer are
pneumatically-operable valves. That is, each valve is operable to
open and close a fluidic channel in which the valve is located, and
this valve is actuated by applying a particular pressure to a
pneumatic control line coupled to the valve. The pneumatic control
lines are coupled to the pneumatic interface 144, to which the
reader has access when the cartridge 100 is inserted therein.
Hence, to actuate a given pneumatic valve, the reader merely
applies an appropriate pressure to the pneumatic control line
associated with that valve to open or close the valve.
[0164] The isolation valve 150 and the bypass valve 153 are also
actuated by the reader, but mechanically. Again, each valve is
operable to open and close a fluidic channel in which the valve is
located, but the valve is actuated by applying one or more
mechanical actuators (such as a foot) to the valve.
[0165] The pneumatic layer further comprises two reference holes
154a-b configured to permit an assembly fixture to provide a
reference to facilitate positioning of the layer during
manufacture. When the cartridge is assembled, the reference holes
154a-b in the pneumatic layer align with the reference holes 134a-b
in the blister sub-assembly.
[0166] The pneumatic layer further comprises apertures 155a-c
which, when the cartridge is assembled, line up with apertures
132a-c passing through the substrate 131 of the blister
sub-assembly (through the pneumatic foil, as described below).
[0167] 1.3.6 Pneumatic Foil 113
[0168] FIG. 7 shows the pneumatic foil 113 in more detail. As
explained above, the pneumatic foil 113 is adhered to the upper
surface of the pneumatic layer 114, thereby fluidly sealing
channels, chambers, valves, pumps, bellows and other components
formed therein. Thus, for the most part, the pneumatic foil 113 is
a generally rectangular and planar foil sheet so as to provide an
effective seal. Beneficially, the pneumatic foil 113 is inert such
that is does not react with the reagents which move through the
pneumatic layer 114.
[0169] However, the pneumatic foil 113 does not overlie the entire
pneumatic layer 114. In particular, the pneumatic foil 113 does not
overlie the sample mixing chamber 136 or the waste chamber 137 at
the non-insertion end 106 of the cartridge 100, or the bypass valve
153 at the insertion end 105. Moreover, the pneumatic foil 113
comprises cut-outs 156, 157, such that it does not overlie the
isolation valve 150 or the pneumatic interface 144,
respectively.
[0170] The pneumatic foil 113 further comprises three apertures
158a-c which, when the cartridge 100 is assembled, line up with
apertures 132a-c passing through the substrate 131 of the blister
sub-assembly and 155a-c passing through the pneumatic layer 114.
The apertures 158a-c permit the liquid reagents within the blisters
to pass to the pneumatic layer 114, and thence to the fluidic layer
115 through apertures 155a-c.
[0171] The pneumatic foil 113 comprises two reference holes 159a-b
configured to permit an assembly fixture to provide a reference to
facilitate positioning of the layer during manufacture. When the
cartridge is assembled, the reference holes 159a-b in the pneumatic
foil align with the reference holes in the other layers.
[0172] The pneumatic foil is a composite foil manufactured from a
layer of polyethylene terephthalate, to provide strength, with a
layer of polypropylene on top to provide an inert material for
contacting the liquid sample and buffers, and also to enable the
foil to be heat sealed to the pneumatic layer (also manufactured
from polypropylene.
[0173] 1.3.7 Fluidic Layer 115
[0174] FIGS. 8A and 8B show the fluidic layer 115 in more detail.
FIG. 8A is a top view of the pneumatic layer and FIG. 8B is a
bottom view. The fluidic layer 115 is comprised of a rigid plastic
layer 160. As explained previously, the top side of the fluidic
layer 115 (not shown) is adhered to the bottom side of the
pneumatic layer 113 (see FIG. 5B) such that the various channels,
chambers, valves, pumps, bellows and other components formed by a
combination of the pneumatic and fluidic layers are aligned.
[0175] As with the rigid plastic layer 135 of the pneumatic layer
113, the rigid plastic layer 160 of the fluidic layer 115 has a
plurality of differently-shaped recesses therein and apertures
therethrough. In combination with the pneumatic layer 113 and the
fluidic foil 116, certain recesses within, and/or apertures
through, the rigid plastic layer 160 forms certain components,
including: the sample inlet chamber 136; the capture column 138;
the elution chamber 139; the first and second amplification
chambers 140a-b; and the first to fourth detection chambers 141a-d.
the upstream bellows valve 142; the bellows 143; the pneumatic
interface 144; the downstream bellows valve 145; the wash buffer
inlet valve 146; the wash buffer air inlet valve 146a; the elution
buffer inlet valve 147; the elution buffer air inlet valve 147a;
the waste chamber valve 148; the elution chamber valve 149; the
isolation valve 150; the first and second amplification chamber
inlet valves 151a-b; and first and second amplification chamber
outlet valves 152a-b. An aperture 161 is also provided to give
access to the electrode layer 117.
[0176] Moreover, in combination with the fluidic foil 116 (but not
the pneumatic layer 114), recesses in the fluidic layer 115 also
provides the coarse filter 162, the convoluted mixing channel 163,
and a plurality of channels which, when the cartridge is assembled,
connect the aforementioned components together to enable passage of
the liquid sample and liquid reagents through the cartridge, and
facilitate pneumatic actuation of the valves, pumps, bellows and
other components.
[0177] The fluidic layer comprises two reference holes 164a-b
configured to permit an assembly fixture to provide a reference to
facilitate positioning of the layer during manufacture. When the
cartridge is assembled, the reference holes 164a-b in the fluidic
layer align with the reference holes in the other layers.
[0178] As mentioned above, channels are formed between the
pneumatic interface and the various valve and bellows described
above. In the exemplary cartridge, the pneumatic interface
comprises 11 ports which are connected to the various components as
follows. [0179] Port 1: bellows [0180] Port 2: upstream bellows
valve [0181] first and second amplification chamber inlet valves
[0182] first and second amplification chamber outlet valves [0183]
Port 3: downstream bellows valve [0184] Port 4: wash buffer inlet
valve [0185] Port 5: wash buffer air inlet [0186] Port 6: wash
buffer air inlet valve [0187] elution buffer air inlet valve [0188]
Port 7: elution buffer air inlet [0189] Port 8: elution buffer
inlet valve [0190] Port 9: reference pressure line [0191] Port 10:
elution chamber valve [0192] Port 11: waste chamber valve
[0193] It will be understood that whilst various inventive aspects
of the exemplary cartridge may be implemented using specific ones
of the connections listed above (in particular, the first and
second amplification chamber inlet and outlet valves being
connected to a single port; and the wash and elution buffer air
inlets being connected to a single port); the precise configuration
listed above is not essential.
[0194] 1.3.8 Fluidic Foil
[0195] FIG. 9 shows the fluidic foil 116 in more detail. As
explained above, the fluidic foil 116 is adhered to the lower
surface of the fluidic layer 115, thereby fluidly sealing channels,
chambers, valves, pumps, bellows and other components formed
therein. Thus, for the most part, the fluidic foil 116 is a
generally rectangular and planar foil sheet so as to provide an
effective seal. Beneficially, the foil 116 is inert such that is
does not react with the reagents which move in the pneumatic
layer.
[0196] However, the fluidic foil 116 does not overlie the entire
fluidic layer 115. In particular, the fluidic foil 116 does not
overlie the detection chambers 141a-d at the insertion end 105.
[0197] The fluidic foil 116 comprises two reference holes 165a-b
configured to permit an assembly fixture to provide a reference to
facilitate positioning of the layer during manufacture. When the
cartridge is assembled, the reference holes 165a-b in the fluidic
foil align with the reference holes in the other layers.
[0198] The fluidic foil is a composite foil manufactured from a
layer of polyethylene terephthalate, to provide strength, with a
layer of polypropylene on top to provide an inert material for
contacting the liquid sample and buffers, and also to enable the
foil to be heat sealed to the fluidic layer (also manufactured from
polypropylene.
[0199] 1.3.9 Electrode Layer 117
[0200] Finally, FIG. 10 shows the electrode layer 117 in more
detail. As explained above, the electrode layer 117 is adhered to
the fluidic layer 115. The electrode layer 117 comprises four sets
of detection electrodes 166a-d. Each set of detection electrodes
166a-d comprises first to third electrical contacts 168a-d which
couple with corresponding electrical contacts in the reader when
the cartridge is inserted therein. Preferably, the electrical
contacts are made of silver to optimise the electrical connection.
Preferably electrodes which are silver plated with silver chloride
are used to ensure a the optimal galvanic behaviour.
[0201] Each set of detection electrodes 166a-d comprises a working
electrode 169a-d; a counter electrode 170a-d and a reference
electrode 171a-d. Each of the electrodes is coupled to a respective
electrical contact. Each set of detection electrodes 166a-d also
comprises a dielectric 172a-d covering the interface between the
electrodes and the respective electrical contacts.
[0202] A skilled person understands that electrochemical signalling
may be used to indicate the presence of genetic or immuno targets.
In the exemplary cartridge this process is performed in the first
to fourth detections chambers 141a-d which are optimised to provide
the electrochemical test interface.
[0203] The electrodes 166a-d are arranged such that a liquid sample
within the first to fourth detection chambers 141a-d comes into
contact with the first to fourth sets of electrodes 166a-d. In the
detection chambers, some compounds in the fluid sample (referred to
as the `electrolyte`) have a natural tendency to migrate to
electrodes and swap electrons. This galvanic effect is how
batteries work.
[0204] All combinations of soluble compounds have some
electrochemical activity, and the rate at which this activity
occurs (i.e. the amount of charge exchanged) is determined by
exactly what those compounds are. Hence, it is possible to measure
the presence of different analytes in the liquid sample, by
searching for characteristic features of their redox
electrochemistry.
[0205] In the exemplary cartridge, the current required to maintain
a given redox state in the detection chambers 141a-d is monitored
at different redox states. Current is supplied through the
electrolyte from the working electrodes 169a-d to counter
electrodes 170a-d.
[0206] The reference electrodes 171a-d also contact the
electrolyte. Voltages are declared with respect to this reference
electrode because its voltage is largely independent of the redox
conditions and this therefore means that it is only the redox state
of the chemistry at the control electrode that is being
measured.
[0207] A voltage sweep is applied between the working electrodes
169a-d and counter electrodes 170a-d by the reader, which generates
the characteristic range of redox conditions. The current passing
between the working electrodes 169a-d and the counter electrodes
170a-d is then measured to obtain the test results. The voltage
sweep is a slowly incrementing set of voltages applied between the
electrodes. Preferably the sweep is from about -0.7 volts to about
+1 volts relative to the reference electrode. The voltage is
applied in consecutive incrementing pulses having a pulse
modulation amplitude of between 30 and 80 millivolts (preferably
between 40 and 60 millivolts; more preferably 50 millivolts).
Preferably the step increment from one pulse to the next is between
1 and 5 millivolts (preferably between 2 and 4 millivolts; more
preferably 3 millivolts). By applying these voltages across the
electrodes, current measurements in the scale of 100s of nano amps
may be obtained.
[0208] The particular arrangement of detection electrodes
illustrated in FIG. 10 may itself form an isolated inventive aspect
of the cartridge. Conventionally, the counter electrode in a
potentiostat is larger than the working electrode to provide an
ample supply of surplus electrons. However, it has been found that
reversing this convention surprisingly offers better results for
the exemplary cartridge. For the electrochemistry performed on the
liquid sample described above in the exemplary cartridge, it is
found that having a working electrode which is larger than the
counter electrode provides larger signals and improved results by
way of increased sensitivity. In other words, having a current flow
from a relatively large working electrode to a relatively small
counter electrode offers improvements over the conventional
arrangement.
[0209] Preferably each working electrodes 169a-d is formed in a
U-shape and each counter electrode 170a-d is formed in a straight
elongate shape between the two prongs of the respective U-shaped
working electrode.
[0210] The method operation of the exemplary cartridge introduced
above will now be briefly explained.
[0211] 1.4 Method of Operation of the Exemplary Cartridge
[0212] 1.4.1 the Front End
[0213] As described above, a fluid sample (such as a urine sample)
is introduced into the sample mixing chamber 10 using a pipette. A
portion of the sample passes to the sample indicator 12 to show
that a sample is present in the sample mixing chamber.
[0214] Once the cartridge 100 with a sample in the mixing chamber
10 is inserted into a reader, and the reader is activated, the test
may commence. Firstly, the reader will apply a mechanical actuator
(such as a foot) to collapse the lysis buffer blister 14. In doing
so, the lysis buffer will be expelled into the sample mixing
chamber 10 where it will mix with the sample.
[0215] The bellows 20 and its valves 22a-b then moves the liquid
sample and lysis buffer back and forth into the sample mixing
chamber 10 so as to mix the lysis and sample and to rehydrate the
internal control. Following the mixing step, incubation of the
sample and lysis buffer occurs to allow cell lysis to take
place.
[0216] The bellows 20 and its valves 22a-b will then commence
operation to pump the sample from the sample mixing chamber 10,
into the main channel 16, through the coarse filter 18 and toward
the capture column 24. Within the capture column 24 nucleic acids
are specifically bound to a filter in the capture column on the
basis of their size and charge. The unwanted liquid sample passes
through to the waste chamber 38.
[0217] Once the unwanted liquid sample has passed to the waste
chamber 38, leaving the nucleic acids bound to the capture column
24, the reader applies a mechanical actuator (such as a foot) to
collapse the wash buffer blister 30. In doing so, the wash buffer
will be expelled into the first branch channel 26, and thence into
the main channel 16. Again, the bellows 20 and its valves 22a-b
will commence operation to pump the wash buffer through the main
channel 16 and through the capture column 24 to wash any remaining
unwanted cell debris and other cellular components out of the
capture column with the wash buffer through to the waste chamber
38, or else the wash buffer will be flushed into the waste chambers
using air from the wash and/or elution buffer air inlets.
[0218] Once the wash sample has passed to the waste chamber 38,
leaving only the bound and purified nucleic acids in the capture
column 24, the reader applies a mechanical actuator (such as a
foot) to collapse the elution buffer blister 32. In doing so, the
elution buffer will be expelled into the second branch channel 28,
and thence into the main channel 16. Again, the bellows 20 and its
valves 22a-b will commence operation to pump the elution buffer
through the main channel 16 and through the capture column 24 to
elute the nucleic acids from the capture column, or else the
elution buffer will be flushed into the capture column using air
from the wash and/or elution buffer air inlets. The prepared liquid
sample then passes through to the elution chamber 46; again, either
by being pumped or flushed as described above.
[0219] The sample settles in the elution chamber 46 allowing
bubbles to disperse before entering the amplification chambers.
[0220] 1.4.2 the Back End
[0221] The bellows 20 and its valves 22a-b will then commence
operation to pump the liquid sample from the elution chamber 46,
through the isolation valve 59, through the mixing channel 52 and
into the amplification chambers 56a-b, or else the sample will be
flushed into the amplification chambers using air from the wash
and/or elution buffer air inlets. In the nucleic acid amplification
chambers 56a-d the nucleic acid of interest, if present, is
amplified such that it is present at a detectable level. The
control nucleic acid is also amplified such that it is present at a
detectable level. As mentioned above, any nucleic acid
amplification method may be used. Where PCR is used, primers
specifically hybridise to the nucleic acid of interest and are
extended by a thermostable polymerase such as Taq polymerase via
the addition of dNTPs to the 3' end of each of the primers. Any
excess liquid sample may be removed from the fluid pathway through
the bypass channels 68.
[0222] The bellows 20 and its valves 22a-b will then commence
operation to pump the liquid sample from the amplification chambers
56a-b and into the detection chambers 62a-d, or else the sample
will be flushed into the detection chambers using air from the wash
and/or elution buffer air inlets. In the detection chambers, the
target probe specifically hybridises to the target amplified
nucleic acid of interest and the control probe specifically
hybridises to the amplified control nucleic acid. The nuclease
hydrolyses the target and control probes following hybridisation of
the probes to the amplified nucleic acid. The hydrolysis of the
target and control probes frees the labels from the probes causing
a detectable change in the signal from the labels to occur.
[0223] Once the liquid sample occupies the detection chambers, the
reader applies a mechanical actuator to the isolation valve 50 to
close the valve and isolate the liquid sample in the back end of
the device.
[0224] The electrodes provide a potential difference across the at
least one detection chamber. Depending on the state of the label
(i.e. whether it is attached to the full length probe or the probe
has been hydrolysed and it is free or attached to a single
nucleotide or short part of the probe), the current that is able to
flow through the detection chamber will differ. The electrodes
therefore allow detection by the reader of the change in the signal
from the label which results from hydrolysis of hybridised
probe.
[0225] The present invention will now be described with reference
to FIGS. 16 to 20.
[0226] 2. Handling the Liquid Sample in the Back End
[0227] The following section describes the present invention in
more detail with reference to FIGS. 16 to 20. The invention may be
implemented in the exemplary fluidic cartridge described above,
specifically in the back end of the cartridge, downstream of the
isolation valve. However, it will be appreciated that the present
invention has a number of advantages which may be applicable in
circumstances other than the exemplary fluidic cartridge described
above.
[0228] 2.1 Metering the Liquid Sample
[0229] FIG. 16 shows a first embodiment of a valve system according
to the invention for metering a liquid sample. The valve system
C100 comprises a fluid pathway C110 for passing fluid from an
upstream end to a downstream end, a sample processing chamber C102
within the fluid pathway, having an inlet valve C101 upstream of
the sample processing chamber C102 and an outlet valve C103
downstream of the sample processing chamber C102. The sample
processing chamber may, for example, be a nucleic acid
amplification chamber 58a-b described above in respect of the
exemplary cartridge, although other chambers are also possible. A
downstream sample processing region may be provided at the
downstream end of the fluid pathway. In the exemplary cartridge,
the downstream sample processing region may be target chamber C104,
which is located along the fluid pathway downstream of the outlet
valve C103. Again, the target chamber may, for example, be a
detection chamber 64a-d described above in respect of the exemplary
cartridge, although other chambers are also possible. Irrespective
of the purposes to which the sample processing chamber and target
chamber are put, the target chamber is a chamber to which a volume
of a liquid sample is to be delivered, once the sample has passed
through the processing chamber.
[0230] A bypass channel C105 is coupled to the fluid pathway at a
junction between the outlet valve C103 and the target chamber C104.
The purpose of the bypass channel C105 is to permit excess liquid
sample, which should be prevented from entering the target chamber,
to be removed from the fluid pathway, as described in more detail
below.
[0231] Inlet valve C101 and outlet valve C103 may be
pneumatically-actuated valves formed in the pneumatic and fluidic
layers of the exemplary cartridge, for example. A diagram of an
exemplary pneumatically-actuated valve is shown in FIG. 17. A valve
cavity C201 may be formed in a single polymer layer or in a
plurality of layers, such as the housing layer 111, pneumatic layer
114, and fluidic layer 115 of the exemplary cartridge described
above. A flexible valve membrane C202 is formed within the valve
cavity C201 to define a valve chamber C203 between the valve
membrane C202 and the valve cavity C201. The membrane may be
overmoulded onto the pneumatic layer, as explained above.
[0232] The valve chamber C203 has a first opening C204 and a second
opening C205, each connected to a channel; either the bypass
channel C105, a channel which forms part of the main pathway C110,
or any other channel. The flexible membrane C202 is movable between
a closed position (FIG. 17a), in which the flexible membrane C202
seals against the first and second openings C204, C205 to prevent
fluid flow through the channel or pathway, and an open position
(FIG. 17b), in which the flexible membrane C202 is spaced apart
from the first and second openings C204, C205 to permit fluid to
flow through the channel or pathway.
[0233] The valve C200 further comprises a passageway C206 having an
opening into the valve cavity C201. The opening of passageway C206
is separated from the first and second openings C204, C205 by the
flexible membrane C202. The passageway C206 serves as an actuation
channel to move the flexible membrane between its open and closed
positions actuate the valve. Preferably, under atmospheric
pressure, valve membrane C202 is sealed against first and second
openings C204, C205 and the valve is closed. Conversely, when a
vacuum or gauge pressure is applied via the fluid passageway C206,
the pressure within the valve cavity C201 reduces below that in the
channel of fluid pathway C110 and the flexible membrane C202 is
brought into the open position. Actuation passageway C206 may be
connected to a port on pneumatic interface through which the vacuum
or gauge pressure may be applied.
[0234] Referring back to FIG. 16, it will be understood that the
inlet and outlet valves C101, C103 may each be configured in
accordance with the valve shown in FIGS. 17a-b. Preferably, the
actuation passageways of the inlet and the outlet valves C101, C103
are coupled to a single port on the pneumatic interface to permit
substantially simultaneous actuation of the inlet and outlet valves
C101, C103. To improve the accuracy of the simultaneous actuation,
the actuation passageways from the valve to the pneumatic interface
may be the same length and the total volume of the passageways and
valve cavities are equal. This ensures that upon application of a
gauge pressure to the actuation passageways via the port on the
pneumatic interface (not shown), inlet valve and outlet valves
C101, C103 will be opened and closed simultaneously. To improve the
speed of actuation, the inlet valve and outlet valves C101, C103
may be provided with abutments, as described in more detail
below.
[0235] The arrangement described above permits a precise volume of
liquid sample to be delivered from the sample processing chamber
C102 to the target chamber C104, as will now be explained. A liquid
sample is first introduced through fluid pathway C110. The sample
passes downstream, through the open inlet valve C101 and into the
sample processing chamber C102. When the sample processing chamber
is full, the liquid sample passes further downstream, through the
open outlet valve C103 such that at least a portion of the liquid
sample is downstream of the outlet valve C103.
[0236] At this point, at least outlet valve C103 (but preferably
both the inlet valve C101 and outlet valve C103) is closed. This
ensures that a fixed and predetermined volume of liquid sample is
contained between the inlet valve C101 and outlet valve C103.
[0237] Once the outlet valve C103 (or the inlet valve C101 and the
outlet valve C103) is closed, excess liquid sample downstream of
the outlet valve may be removed from the fluid pathway via the
bypass channel C105. A preferred arrangement for removing the
excess liquid sample is described below, but any means will do for
the purpose of describing the present embodiment of the invention.
For instance, a vacuum may be applied to the bypass channel C105 to
remove the liquid sample and an appropriate re-pressurising system
provided to ensure that the fluid pathway returns to its normal
operating pressure.
[0238] Once the surplus liquid sample has been removed from the
fluid pathway downstream of the outlet valve C103 via the bypass
channel C105, a fixed and predetermined volume of liquid sample
exists between the inlet valve C101 and the target chamber C104.
Thus, the fixed and predetermined volume of liquid sample may be
delivered to the target chamber C104 simply by opening the outlet
valve C103 and passing the liquid from the sample processing
chamber to the target chamber by any convenient process, such as
described above.
[0239] As will now be explained, when the valve system described
above in connection with FIG. 16 is used in the exemplary cartridge
fluidic cartridge, a metering system C300 is established for
delivering a well-defined volume of processed sample to detection
chambers C306.
[0240] Referring now to FIG. 18, in the exemplary cartridge
described above, two fluid pathways are provided. Of course, more
or fewer pathways may be provided depending on the preferred
implementation.
[0241] First fluid pathway C310a comprises a first sample
processing chamber C302a, a first inlet valve C301a upstream of the
first sample processing chamber C302a, a first outlet valve C303a
downstream of the first sample processing chamber C302a and first
and second detection chambers C304a1, C304a2 branching from the
main channel of the first fluid pathway, downstream of the first
outlet valve C303a. Again, more or fewer detection chambers may be
provided per fluid pathway, depending on the preferred
implementation.
[0242] Likewise, second fluid pathway C310b comprises a second
sample processing chamber C302b, a second inlet valve C301b
upstream of the second sample processing chamber C302b, a second
outlet valve C303b downstream of the second sample processing
chamber C302b and third and fourth detection chambers C304b1,
C304b2, branching from the main channel of the second fluid pathway
C310b, downstream of the second outlet valve C303b.
[0243] First and second bypass channels C305a C305b are coupled to
the first and second fluid pathways C310a, C310b respectively
between the outlet valves C303 and the detection chambers C304. If
more or fewer fluid pathways were provided, it will be appreciated
that a corresponding number of bypass channels may be connected
thereto at a corresponding number of junctions.
[0244] Although in the embodiment illustrated in FIG. 18, the ratio
of sample processing chambers to target chambers is 1:2, it will be
appreciated that the ratio may be 1:1, 1:3 or 1:n such that there
are n target chambers branching from the main channel of each fluid
pathway.
[0245] Referring still to FIG. 18, in an exemplary embodiment of
the valve system of the present invention, there is further
provided a control valve (or `bypass` valve) C315 to which the
first and second bypass channels are coupled, first and second
fluid pathways stemming from the isolation valve 50, and first,
second, third and fourth gas springs C306, downstream of the first,
second, third and fourth target chambers C304 respectively. The
bypass valve C315 is a valve which is used to control the movement
of a liquid sample within the back end, as described in more detail
below. The liquid sample enters the back end of the fluidic
cartridge through isolation valve 50 and then enters amplification
chambers C302a, C302b. Gas springs 306a1-2 and 306b1-2 are blind
bores (that is, dead-ends in the channels) which contain a
compressible gas. The compressible gas is compressed as a fluid is
pushed into the channel in which the gas spring is located, and the
compressible gas thus exerts a force against the fluid in the
channel in a direction opposite to that from in which it is
introduced.
[0246] An implementation of valve system C300 will now be explained
with reference to FIGS. 18 and 19. A liquid sample is introduced
into the back end of the exemplary cartridge via isolation valve 50
as described above. As liquid sample is introduced, first and
second inlet valves C301a-b and first and second outlet valves
C303a-b are open, and bypass valve C305 is closed. As the liquid
sample is passed along the first and second fluid pathways C310a-b,
the volume between the dead-end of gas springs C306a1-2, C306b1-2
and the fluid sample in the fluid pathways reduces. Since bypass
valve C315 is closed, no air can escape downstream of the advancing
fluid sample, and gas springs C306a1-2, C306b1-2 become
pressurised. The liquid sample continues to be advanced along first
and second fluid pathways C310a-b until it passes the outlet valves
C303a-b, at which point it is known that a surplus of fluid has
been delivered to sample processing chambers C302a-b. Once a
surplus of liquid sample has been delivered and the sample
processing chambers C302a-b are full, inlet and outlet valves
C301a-b and C302a-b are closed.
[0247] Once the inlet and outlet valves C301a-b and C302a-b are
closed, the sample is processed in the sample processing chambers
C302a-b. In the exemplary cartridge described above, it is
envisaged that PCR amplification will be performed on the sample.
Once the inlet and outlet valves C301a-b and C302a-b are closed,
and whilst the liquid sample is being processed in sample
processing chambers C302a-b, bypass valve C315 is opened, whilst
the first and second inlet and outlet valves C301a-b and C302a-b
remain closed. The bypass valve may be opened whilst the liquid
sample is being processed, or after or before the liquid sample is
processed. When bypass valve C315 is opened, first and second
bypass channels C305a-b are permitted return to atmospheric
pressure by virtue of the bypass valve being vented to atmosphere
in any convenient manner. Since the pressure in bypass channels
C305a-b is now less than the pressure in the pressurised gas
springs C306a1-2, C306b1-2, the surplus fluid in the fluid pathways
C310a-b is forced out of the pathways and into the bypass channels
C305a-b by the force exerted from the gas springs C306a1-2,
C306b1-2. To ensure that substantially all the surplus liquid
sample is forced out of the pathways, the bypass channels C305a-b
are located immediately adjacent the outlet valves 303a-b to
prevent dead-legs from forming between the outlet valves 303a-b and
the junctions at which the bypass channels 306a-b join the fluid
pathways.
[0248] Once the surplus fluid sample has been drawn into bypass
channels C305a-b, first and second inlet and outlet valves C301a-b
and C302a-b are opened and the processed sample is advanced along
the first and second fluid pathways and delivered to the detection
chambers C304a1-2, C304b1-2.
[0249] Steps of the method described above are set out in FIG.
19.
[0250] 2.2. Evacuating Excess Liquid Sample
[0251] In the above discussion of a valve system for metering a
liquid sample in a sample chamber, an example was given of a
mechanism for expelling a surplus liquid sample using gas springs.
This novel mechanism for expelling a surplus liquid sample need not
only be used in conjunction with a sample processing chamber
bounded by inlet and outlet valves, and could instead be used to
expel a surplus liquid sample from a main fluid pathway downstream
of an outlet valve of any subsystem or sample processing region in
a fluidic cartridge, to ensure that only the contents remaining
upstream of the outlet valve is passed to the target chamber.
[0252] Thus, FIG. 20 shows a second embodiment of a valve system
according to the invention for expelling a liquid sample from
sub-system such as a sample processing region (not shown). The
valve system C500 comprises a fluid pathway C510 for passing fluid
from an upstream end to a downstream end, an outlet valve C503
downstream of the sample processing region (not shown), and a
target chamber C504 located along the fluid pathway downstream of
the outlet valve C503. The target chamber may, for example, be a
detection chamber 64a-d described above in respect of the exemplary
cartridge, although other chambers are also possible, depending on
the particular sample processing region. Irrespective of the
purpose to which the target chamber is put, the target chamber is a
chamber to which a volume of a liquid sample is to be delivered,
once the sample has exited the sample processing region.
[0253] A bypass channel C505 is coupled to the fluid pathway
between the outlet valve C503 and the target chamber C504. The
purpose of the bypass channel C505 is to permit excess liquid
sample, which should be prevented from entering the target chamber
C504, to be removed from the fluid pathway, as described in more
detail below.
[0254] A gas spring C506 is provided downstream of the target
chamber C504. As explained above, the gas spring C506 is a blind
bore (that is, a dead-end in the channel) which contains a
compressible gas. The compressible gas is compressed as a fluid is
pushed into the channel in which the gas spring is located, and the
compressible gas thus exerts a force against the fluid in the
channel. A bypass valve C515 is also provided within the bypass
channel. The bypass valve is a valve which is used to control the
movement of a liquid sample, as described below
[0255] A liquid sample passes from the sample processing region
(not shown) and downstream of the open outlet valve C503. As liquid
exits the sample processing region, the outlet valve C503 is open
and the bypass valve C515 is closed. As the liquid sample is passed
along the fluid pathway C510, the volume between the dead-end of
the gas spring C506 and fluid sample reduces. Since bypass valve
C515 is closed, no air can escape downstream of the advancing fluid
sample, and gas spring C506 becomes pressurised. The liquid sample
continues to be advanced along the fluid pathway C510 until it
passes the outlet valve C503. Once this happens, outlet valve C503
is closed.
[0256] Once outlet valve is closed, bypass valve C515 is opened,
whilst the outlet valve remains closed. When bypass valve C515 is
opened, the bypass channel C505 is permitted return to atmospheric
pressure, again by virtue of the bypass valve being vented to
atmosphere in any convenient manner. Since the pressure in bypass
channel C505 is now less than the pressure in the pressurised gas
springs, the surplus fluid in the fluid pathway is forced out of
the pathway and into the bypass channel C505 by the force exerted
from the gas spring 506. To ensure that substantially all the
surplus liquid sample is forced out of the pathway, the bypass
channel is located immediately adjacent the outlet valve C503 to
prevent a dead-leg from forming between the outlet valve 503 and
the junction at which the bypass channel C506 joins the fluid
pathway.
[0257] Once the surplus fluid sample has been expelled into bypass
channel C505, the outlet valve C503 is opened and processed sample
is advanced along the fluid pathway and delivered to the detection
chamber C504.
[0258] It will be recognised that the embodiment discussed in
connection with FIG. 20 may be implemented with any number of fluid
pathways, any number of target chambers, and any number of gas
springs. It will also be recognised that the embodiment discussed
in FIG. 20 may be implemented in the exemplary fluidic cartridge in
the manner described above in connection with FIGS. 18 and 19.
[0259] Once the excess liquid sample has been removed from the
fluid pathway and passed into the bypass valve, it may be prevented
from returning into the fluid pathway by any convenient means. For
example, in the exemplary fluid cartridge, the isolation valve and
bypass valve may be configured to reduce the pressure in the back
end of the cartridge, and preferably develop a negative fluid
pressure in the back end of the cartridge, thereby sucking the
excess liquid sample toward the bypass valve and preventing it from
returning toward the fluid pathway.
[0260] The use of gas springs in the embodiments described above in
connection with FIGS. 18 to 20 is particularly advantageous because
it permits equal quantities of processed sample to be delivered to
the target chambers even when local imbalances in pressures (such
as those caused by thermo cycling in a nucleic acid amplification
process, for example) may make such precise delivery difficult. By
venting gas springs C306 and target chambers C304 through bypass
channels C305 when the bypass valve is open, and allowing the
target chambers to equalise, the pressure within the target
chambers can remain equal and ensure delivery of equal quantities
of liquid sample.
[0261] Referring back to the implementation of the valve systems of
the present invention in the exemplary cartridge (see FIGS. 18 and
19), it is preferred that the combined volume of the plurality of
detection chambers branching from each sample processing chamber is
approximately half of the volume of sample processing chamber
itself. This is because as processed sample from sample processing
chamber C302 is advanced, unprocessed sample from upstream of the
sample processing chamber is also passed along each fluid pathways
C310 and mixes with the processed fluid downstream of the sample
processing chamber C302. By ensuring that there is twice as much
processed fluid available than the combined capacity of the
plurality of detection chambers, only the undiluted processed fluid
will be advanced into detection chambers C306. Of course, this
ratio is merely preferred, and in reality any ratio wherein the
volume of the sample processing chamber is larger than the combined
volumes of the target chambers would work.
[0262] 2.3 the Bypass Valve and Valve System
[0263] The present invention may be implemented together with a
preferred bypass valve, which will now be described, to achieve an
improved back end region in a fluidic cartridge, such as the
exemplary fluidic cartridge described above. The bypass valve may
be the bypass valve 68 of the exemplary fluidic cartridge described
above. However, it will be appreciated that the valve of the
present invention has a number of advantages, which may be
applicable in circumstances other than the exemplary fluidic
cartridge.
[0264] FIG. 21 shows a first embodiment of a valve D100. The valve
D100 comprises a valve cavity D101 and a flexible valve membrane
D105 provided within valve cavity D101. The valve cavity D101 may
be formed in a single polymer layer or may be comprised of a
plurality of layers, such as the housing 111, pneumatic layer 114,
and fluidic layer 115 of the exemplary cartridge 100. The valve
membrane D105 is formed within the valve cavity D101 and may be
over-moulded onto the pneumatic layer, as explained above.
[0265] The valve cavity D101 has side walls D130, a floor D132 and
is open at the top. The valve cavity D101 comprises first and
second openings D102, D103 in the floor D132 connected to first and
second passageways, D112, D113 formed in the fluidic layer 115.
First and second openings D102, D103 may be located on first and
second raised portions of valve cavity D101 to form first and
second valve seats.
[0266] Between the flexible membrane D105 and the floor D132 of the
valve cavity D101, a valve chamber D115 is defined. The valve
chamber is therefore fluidly connected to the first and second
openings D102, D103.
[0267] Valve membrane D105 comprises a first valve membrane portion
D122 and a second valve membrane portion D123.
[0268] The first valve membrane portion D122 is movable between an
open position, in which it is spaced apart from the first opening
D102 and permits fluid to flow between the first passageway D112
and the valve chamber D115, and a closed position, in which it
seals against the first opening D102 and prevents any fluid flow
between the first passageway D112 and the valve chamber D115.
[0269] Similarly, the second valve membrane portion D123 is movable
between an open position in which it is spaced apart from the
second opening D103 and permits fluid to flow between the second
passageway D113 and the valve chamber D115, and a closed position,
in which it seals against the second opening D102 and prevents any
fluid flow between the second passageway D113 and the valve chamber
D115.
[0270] It will therefore be appreciated that the valve chamber D115
has a first volume, V1, when the first and second valve portions
are in their open positions; a second volume, V2, when one of the
first and second valve portions is in its open position and the
other is in its closed position; and a third volume, V3, when the
first and second valve positions are in their closed positions. It
will be appreciated that V1>V2>V3. Volume V3 is ideally as
small as possible, and may be substantially zero.
[0271] The first and second valve membrane portions D122, D123 are
actuatable independently of one another. For instance, when the
valve is used in the exemplary cartridge and when the cartridge is
inserted into a reader, the reader may apply first and second
mechanical actuators, such as feet, to actuate the first and second
valve membrane portions D122 and D123 independently. This is
advantageous in a sealed system, such as the back end of the
exemplary cartridge where there is a critical pressure to be
maintained on one side of the valve. In this case the valve seat
corresponding to the first and second channels can be actuated
first while keeping the valve chamber open to the bypass channel,
to avoid pressurising the first and second channels or displacing
the liquid therein.
[0272] Referring now to FIG. 22, a second embodiment of a valve
D200 is illustrated. The second embodiment is identical to the
first (and like reference numerals refer to similar features),
except that a third opening D204 is provided in the floor of the
valve cavity in addition to the first and second openings D202,
D203. The third opening D204 is connected to a third passageway
D214 and is adapted to be sealed by the second valve membrane
portion D223, to prevent fluid moving between the third passageway
D214 and the valve chamber D250.
[0273] In the embodiment shown in FIG. 22, second and third
openings D203, D204 are located on second and third raised portions
D243, D244. However, it will be appreciated that second and third
opening may be located on a single raised portion D343 as shown in
FIG. 23, or that they may be located on a region substantially
flush with the rest of the valve cavity floor.
[0274] As shown, the second and third openings D203, D204 are
spaced apart by distance b. First opening D202 is spaced apart from
the second opening D203 by distance a. The distance a between first
and second openings D202, D203 is greater than the distance b
between second and third openings D203, D204. This is convenient to
enable the second and third openings D203, D204 to be sealed by the
second membrane portion D223 and the first opening D202 to be
sealed by the first membrane portion D222.
[0275] Although in the first and second embodiments illustrated in
FIGS. 21 and 22, the valve is shown as having two or three
openings, it will be appreciated that four or more openings may be
provided. The openings may be grouped in any manner so as to be
sealed by the first membrane portion or the second membrane
portion, depending on the preferred implementation. It will also be
appreciated that, although in the embodiments shown in the
drawings, the valve membrane is shown to have first and second
valve membrane portions, it possible that the valve membrane may
have three or more portions, each adapted to seal one or more
openings and each adapted to be independently actuatable.
[0276] As described above, the first valve membrane portion D222
and the second valve membrane portion D223 may be mechanically
actuated by first and second mechanical actuators D232, D233 which
could, for instance, be provided in a reader (not shown). The first
mechanical actuator D232 is configured to be movable from a first
position in which it is spaced apart from the first valve membrane
portion D222 and a second position in which it presses first valve
membrane portion against opening D202, thereby sealing the opening.
Similarly the second mechanical actuator D232 is configured to be
movable from a first position in which it is spaced apart from the
second valve membrane portion D223, to a second position in which
it presses second valve membrane portion D233 against the second
opening and third openings D203, D204.
[0277] The valve may be configured such that the mechanical
actuators D232 and D233 may contact substantially all of the valve
membrane D205. Alternatively, the valve may be configured such that
the mechanical actuators D232 and D233 may only contact a portion
of valve membrane D105.
[0278] Referring now to FIGS. 23 and 24, it can be seen that by
positioning the second and third openings relatively close
together, the second valve membrane portion D223 may be actuated to
effectively seal the second and third openings D203, D204 without
requiring a large surface area to contact the valve membrane D205.
In contrast, the relatively large distance between the second and
first openings D202, D203 allows the second portion of valve
membrane D223 to be depressed by the second biasing means without
significantly depressing the first valve membrane portion D222.
[0279] Preferably, valve membrane D205 is formed of resiliently
deformable polymer such that the valve is biased into the first
position. Preferably, the valve membrane D205 has a thickness of at
least 0.25 mm, most preferably a thickness of around 1 mm. This
ensures that the valve membrane is thick enough to provide
compliance for an effective seal over the openings. By moving the
biasing means D232, D233 from the second position to the first
position, biasing means D232, D233 no longer press valve membrane
D105 against openings D202, D203, D204 and the valve returns to the
open position.
[0280] An implementation of the valve D200 will be explained with
reference to FIG. 25. In particular valve D200 is used as a bypass
valve 68 in the back end of the exemplary fluidic cartridge 100
discussed above.
[0281] FIG. 25 shows following features of the exemplary cartridge
100: the elution branch 16b of the main channel 16; the isolation
valve 50; the mixing channel 52; the first and second PCR channels
54a, 54b and the first and second bypass channels 66a, 66b. Certain
features present in the exemplary cartridge 100 are omitted from
FIG. 15 for clarity.
[0282] The network of channels and valves referred to in the
previous paragraph form a valve system D500; namely part of the
back end of the exemplary fluidic device. It will be appreciated
that invention may be implemented in other valve systems and with
other networks of channels, depending on the preferred
implementation. In particular, it will be appreciated that the
system in FIG. 25 may be combined with the features described under
sections 2.1 and 2.2 above.
[0283] As illustrated in FIG. 25, the first and second bypass
channels 66a, 66b are respectively connected to the second and
third openings D203, D204 of the bypass valve D200. The first
opening 202 and first passageway D212 are coupled to the elution
branch 16b of the main channel 16 downstream of the isolation
valve.
[0284] As described above, the back end of the exemplary fluid
cartridge forms a closed system when the isolation valve 50 is
closed. Hence, a first advantage of using the bypass valve D200 in
the valve system D500 shown in FIG. 20 is that it can be used to
depressurise the back end after the test is complete. This may
occur as follows. Once the liquid sample has been pumped into the
detection chambers (not shown) of the exemplary cartridge 100, the
isolation valve may be closed to form a closed system in the back
end. However, at a suitable point before the isolation valve 50 is
closed, the first, and preferably second flexible membrane portions
D222, D223 may be pushed by mechanical actuators into their closed
positions, thereby decreasing the volume within the valve chamber
D250.
[0285] When the volume of the valve chamber D250 is below its
maximum (for instance when one or both of the flexible membrane
portions D222, D223 is in its closed position), the isolation valve
may be closed, thereby forming a sealed system in the back end.
Once the isolation valve is closed, the flexible membrane portions
D222, D223 may be returned to their open positions, thereby
increasing the volume of the valve chamber D250 to its maximum.
[0286] In one example, when valve D200 is in the open position and
valve membrane D205 is spaced apart from openings D203, D204, as
shown in FIG. 22, the volume of valve chamber D250 is V.sub.chamber
(open). When valve D200 is in the closed position, as shown in FIG.
24, the volume of valve chamber D250 may be approximately 69 .mu.l,
but could be other volumes, including substantially zero.
[0287] Hence, when valve D200 is the open position, the volume of
the valve system D500 is:
V.sub.open=V.sub.chamber (open)+V.sub.network.
[0288] When valve D200 is in the closed position, the volume of the
valve system D500 is:
V.sub.closed=V.sub.chamber(closed)+V.sub.network.
[0289] It will be appreciated that when the closed volume of the
valve chamber is substantially zero:
V.sub.closed=V.sub.network.
[0290] Providing the isolation valve is open when valve D200 is
closed, an amount of fluid equal to V.sub.chamber
(open)-V.sub.chamber (closed) will be displaced outside the network
of channels (upstream of the isolation valve) and there will be an
amount of fluid equal to V.sub.network V.sub.chamber(closed) left
in the network of channels (downstream of the isolation valve).
[0291] When isolation valve 50 is closed, the system becomes a
closed system and the quantity of fluid in that system is fixed.
When valve D200 is then reopened after the isolation valve has been
closed, the volume of the system returns to V.sub.open. Since
V.sub.open>V.sub.closed, the pressure in the system is reduced,
and, preferably, a negative pressure is created in the system. This
reduction in pressure reduces the risk of leakage of the cartridge.
It will be appreciated that if this reduction in pressure is large
enough, it is possible to create a negative pressure in a system,
even where the system is initially slightly pressurised.
[0292] By closing valve D200 to reduce the volume of the system,
closing the isolation valve to close the system, and then opening
valve D200 to increase the volume of the system, it is possible to
reduce the pressure in the system, and preferably achieve a
negative pressure within the back end of the exemplary cartridge.
Preferably, the change in the volume of valve chamber D250 is large
enough to effect a significant pressure change in the fluidic
network. Although in the embodiments shown in the drawings, valve
chamber is shown to have two or three openings, it will be
appreciated that this method of depressurising a system will work
with any number of openings.
[0293] As described above, the first and second bypass channels
66a, 66b may be used to remove excess fluid sample from the first
and second fluid pathways through the first and second PCR channels
54a, 54b. Thus, the first bypass channel 66a is coupled to the
first fluid pathway in the first PCR channel 54a and the second
bypass channel 66b is coupled to the second fluid pathway in the
second PCR channel 54b.
[0294] At an appropriate point in the test, it is necessary to
close the bypass valve D200. However, when closing the bypass
valve, there is a risk that the pressure change caused by the
membrane sealing against the second and third openings D203, D204
will push fluid in the bypass channels 66a, 66b back toward the PCR
channels 66a, 66b, particularly if fluid is unable to escape
elsewhere in the system. This is undesirable. Hence, a second
advantage of using the bypass valve D200 in the valve system D500
shown in FIG. 25 is that the pressure change causing such backflow
can be mitigated.
[0295] By using a valve D200, a first step of applying a force to
the second valve membrane portion may be carried out to seal the
second valve membrane portion against the second and third openings
of the valve chamber. FIG. 23 shows the valve D200 in an
intermediate position wherein second and third openings are sealed
by second valve membrane portion D223 whilst the first opening D202
remains open. A second step is then performed which comprises
applying a force to the first valve membrane portion D222 to seal
the first valve membrane D222 portion against the first opening
D202 in the valve chamber D250.
[0296] By closing the second and third openings before closing the
first opening in the valve chamber, it is possible to avoid
pressurising the second and third passageways excessively, and in
fact to minimise the back flow into the first and second bypass
channels.
[0297] Although the method described above refers to a valve having
first, second and third openings, it will be appreciated that this
method may be adapted for valves having two or four or more valves
arranged in two groups, wherein the first group of valves is sealed
by the first valve membrane portion, and the second group of valves
may be sealed by the second valve membrane portion. In this
context, it is intended that a group refer to one or more
valves.
[0298] Preferably, in embodiments having four or more openings, the
first valve membrane portion is configured to seal the first
opening and the second valve membrane portion is adapted to seal
any subsequent openings. Additionally, the opening of the valve
chamber may be located on a raised portion of the valve chamber to
create a raised valve seat. Each raised portion may comprise
multiple openings, or each opening may be provided with its own
raised portion. Alternatively some or all of the openings may not
be located on a raised portion.
[0299] Although preferred embodiments of the present invention are
illustrated in the figures, it should be understood that various
alternatives to the embodiments of the invention described herein
can be employed in practicing the invention.
[0300] 3. Additional Isolated Inventive Aspects
[0301] The following is a non-exhaustive list of isolated aspects
of the exemplary cartridge described above which may be claimed.
These aspects are described with reference to FIGS. 11 to 15. The
inclusion of this section does not preclude there being further
aspects of the exemplary cartridge described above which may also
be claimed.
[0302] 3.1 Valves for Minimising Dead Volume
[0303] An advantageous arrangement for a valve in a fluidic
cartridge will now be described, which may form an isolated
inventive aspect.
[0304] Hence, in one aspect, there is provided a valve for a
fluidic cartridge, the valve comprising: [0305] a valve cavity
having first and second openings connected to first and second
passageways, respectively; and [0306] a flexible membrane movable
between a closed position, in which the flexible membrane seals
against the first and second openings to prevent fluid flow between
the first and second passageways, and an open position, in which
the flexible membrane is spaced apart from the first and second
openings to permit fluid to flow between the first and second
passageways; [0307] wherein the a valve cavity comprises a roof and
a floor, the floor comprising said first and second openings; and
further comprising: [0308] an abutment between the flexible
membrane and the roof of the valve cavity, such that the abutment
restricts movement of the membrane in its open position.
[0309] Preferably the abutment is provided on the flexible
membrane, and comprises one or more of a protrusion, a cage, a lip
or a cross structure.
[0310] It is sometimes advantageous to limit the extent to which
the flexible membrane in a valve described herein is able to travel
in its open position. That is, it is desirable to minimise the
distance which the valve membrane moves to its open, and thus
minimise the distance it must travel to close. By minimising this
distance, the dead volume within the valve cavity is reduced,
improving the reactivity of the valve.
[0311] Hence, as shown in more detail in FIG. 11, preferred
embodiments of a valve 300 further comprise an abutment 302. The
abutment of the illustrated example is a cross structure, but in
different embodiments may be a protrusion, cage, lip or similar,
attached to the upper surface of the flexible membrane 304 so as to
contact the roof 306 of the valve cavity and thus limit movement of
the membrane in its open position.
[0312] It should be appreciated that the channels and openings of
the valve are not shown in FIG. 11.
[0313] The abutment is particularly advantageous when filing the
amplification chambers of the exemplary cartridge, because it
reduces the dead-volume in the valve cavity and thus limits the
distance between the bottom surface of the flexible membrane and
the openings in the valve cavity, thereby permitting a more precise
volume of fluid to be metered into the amplification chambers.
[0314] 3.2 Crack Pressure in Valves
[0315] An advantageous arrangement for a valve in a fluidic
cartridge will now be described, which may form an isolated
inventive aspect.
[0316] Hence, in one aspect, there is provided a valve for a
fluidic cartridge, the valve comprising: [0317] a valve cavity
having first and second openings connected to first and second
passageways, respectively; [0318] a flexible membrane within the
valve cavity movable between a closed position, in which the
flexible membrane seals against the first and second openings to
prevent fluid flow between the first and second passageways, and an
open position, in which the flexible membrane is spaced apart from
the first and second openings to permit fluid to flow between the
first and second passageways; wherein [0319] the valve is
configured such that a pressure required in the first passageway to
move the flexible membrane from the closed position to the open
position is higher than a pressure required in the second
passageway to move the flexible membrane from the closed position
to the open position.
[0320] It will be appreciated that within the valve cavity there is
a portion (known as the valve chamber) between the flexible
membrane and the floor. There is also a portion within the valve
cavity on the other side of the flexible membrane to the valve
chamber. This portion will have a volume. The pressure within that
volume may be changed by applying a positive or gauge pressure to
the volume via an actuation channel, for example. The actuation
channel may be connected to a source of positive or gauge pressure
via a pneumatic interface, for example. The pressure within the
volume is known as the actuation pressure. This operation is
described in more detail above.
[0321] In a preferred arrangement, the first and second openings
may be arranged such that fluid in the first passageway acts on the
flexible membrane only over a relatively small cross-sectional area
whereas fluid in the second passageway acts on the flexible
membrane over a larger cross-sectional area, preferably
substantially the whole membrane.
[0322] The effect of this is that the valve is able to withstand a
much greater pressure in the first passageway that in the second
passageway.
[0323] Preferably the valve cavity has a floor comprising the first
and second openings and one or more walls between which the
flexible membrane extends; and wherein the second opening is
coupled to a recess in the floor between the opening and the
flexible membrane, the recess having a larger cross-sectional area
than the opening.
[0324] Preferably the first opening is located centrally within the
floor and the recess extends around the first opening, such that
the second opening is located between the first opening and a wall
of the valve cavity. In a particularly preferred arrangement, the
valve cavity has a circular cross section, and the recess is an
annular recess which surrounds the first opening.
[0325] Preferably the opening of the second fluid passageway is
located adjacent the perimeter of the valve chamber. Preferably the
valve chamber has a diameter of between 2 and 10 mm, preferably
between 3 and 7 mm and more preferably 4 and 6 mm. More preferably,
the second opening is offset by 2 mm from the first opening.
[0326] An exemplary valve is shown in FIG. 12 in its closed
position. The valve 310 may be used in place of any of the valves
of the exemplary fluidic cartridge shown above. The valve comprises
a valve cavity 312 having a flexible membrane 314 overlying a
cavity floor 316 in which first 318 and second 320 apertures are
provided, leading to first 322 and second 324 fluid passageways,
respectively.
[0327] The cavity 312 is formed from a void in a first polymer
layer (preferably the fluidic layer 114 of the exemplary cartridge)
together with a second polymer layer (preferably the second fluidic
layer 115 of the exemplary cartridge).
[0328] The flexible membrane 314 is shown lying across the floor
316 of the cavity such that the valve is shown in its closed
position. The valve is movable from this position to an open
position (where it is spaced from the floor 316 and the apertures
322, 324 to form a valve chamber), as described herein.
[0329] The first opening 318 of the valve is centrally located
within the perimeter of the void formed in the first polymer layer,
and is therefore centrally located in the valve cavity 312. The
second opening 324 of the valve is offset from the first opening
322. The second opening is coupled to an annular recess 326 in the
floor, and thus the cross-sectional area over which the fluid in
the second passageway 324 acts on the flexible membrane 314 is much
larger than the cross-sectional area over which the fluid in the
first passageway 322 acts on the flexible membrane.
[0330] The pressure of a fluid in the first passageway acts on the
flexible membrane only over a relatively small cross-sectional area
of the flexible membrane. Thus, because the pressure of a fluid in
the valve cavity on the other side of the flexible membrane acts
over the whole membrane, it may be lower without allowing the
membrane to move to its open position.
[0331] In contrast, the pressure of a fluid in the second
passageway acts on the flexible membrane over a relatively large
cross-sectional area of the flexible membrane. Since the respective
cross-sectional areas are closer, so too is the pressure in the
second passageway which the flexible membrane is able to withstand
vis-a-vis the pressure in the valve cavity.
[0332] Preferably, the respective cross-sectional areas of the
openings of the fluid passageways allows the membrane to resist
pressures around 2.5 times the actuation pressure on the first,
central, fluid passageway, but only pressures equal to the
actuation pressure (i.e. the pressure in the valve cavity) on the
opening of the second, offset, fluid passageway.
[0333] 3.3 Entry Port Design
[0334] An advantageous arrangement for an entry port on a fluidic
cartridge will now be described, which may form an isolated
inventive aspect.
[0335] Hence, in one aspect, there is provided a fluidic cartridge
for processing a liquid sample, the cartridge having a sample
mixing chamber comprising: [0336] a sample inlet aperture for
introducing a liquid sample into the sample mixing chamber; [0337]
a cage surrounding the inlet aperture and extending into the sample
mixing chamber, the cage further comprising one or more protrusions
extending radially inwardly to abut against a sample delivery
device introduced through the sample inlet.
[0338] The body of the cage may be formed from one or more elongate
bars, or one or more solid walls, depending from the roof of the
sample mixing chamber. If solid walls are provided, there is
preferably an aperture in the lower portion of the walls through
which a liquid sample introduced by the sample delivery device can
pass. Preferably the bars or wall forming the body are tapered to
conform to the nib of a conventional sample delivery device
introduced through the sample inlet.
[0339] Solid walls have the additional advantage that they provide
a barrier to prevent fluid introduced into the mixing chamber from
escaping out of the inlet aperture, which is particularly useful if
the cartridge is turned upside-down during use.
[0340] If the cage is formed from solid walls, the protrusion may
be a ledge extending inwardly from the walls leaving an aperture.
Preferably the protrusion extending from the sides of the inlet
aperture is positioned above the floor of the sample mixing
chamber; more preferably above a liquid fill level of the sample
mixing chamber. This prevents liquid sample from being sucked back
into the sample delivery device once introduced into the mixing
chamber.
[0341] Preferably a vent is provided in the sample mixing chamber
to allow air to escape from the chamber during the introduction of
the sample. This is particularly useful when the inlet aperture is
sealed by the sample delivery device.
[0342] Preferably a guide channel is provided within the sample
mixing chamber (a portion of which is preferably directly
underneath the inlet aperture) to direct the sample introduced by a
sample delivery device into a visual indicator region. An exemplary
visual indicator region is described above in connection with the
exemplary cartridge.
[0343] Preferably a change in refractive index of the visual
indicator region described herein identifies when a sample has been
introduced. The visual indicator region may comprise a narrow fluid
passageway, which becomes filled by the fluid sample via capillary
action. The filling of the narrow fluid passageway changes the
refractive index of the visual indicator region and a colour change
identifies when a sample has been introduced.
[0344] A preferred embodiment of this aspect will now be described
with reference to the exemplary fluidic cartridge. The housing 111
(see FIG. 4) comprises a sample inlet aperture 126 through which a
sample may be introduced into the sample mixing chamber 10 of the
cartridge 100 using a pipette, for example. As shown in more detail
in FIG. 13a, the sample mixing chamber 10 is formed from the
pneumatic layer 114, which has a roof adjacent the housing 111 in
the region of the inlet aperture, and a corresponding inlet
aperture through which a sample may be introduced into the sample
mixing chamber 10.
[0345] The roof of the mixing chamber 10 comprises a cage structure
formed by walls 330 surrounding the inlet aperture 126 which extend
into the sample mixing chamber 10 from the roof, and a ledge 332
extending radially inwardly from the walls 330. The shape of the
cage structure allows a sample delivery device, such as a pipette,
to be located in the correct position in the sample mixing chamber
10, and the ledge 332 prevents the pipette contacting the surfaces
of the sample mixing chamber 10, thereby reducing the risk of
contamination. The walls 330 can be tapered to further increase the
engagement with the pipette.
[0346] Once the sample delivery device is located through the
aperture, the user can dispense the sample. The ledge 332 is
positioned above a nominal liquid fill level (not shown) of the
sample mixing chamber so as to prevent the user from accidentally
sucking the sample back up after dispensing it into the
chamber.
[0347] A vent 334 into the chamber is provided to allow air to
escape in the event that the inlet aperture is sealed by the sample
delivery device.
[0348] A guide 336 is provided within the sample mixing chamber 10,
a portion of which is directly underneath the inlet aperture 126 to
direct the sample introduced by a sample delivery device into a
visual indicator region 338. An exemplary visual indicator region
is described above in connection with the exemplary cartridge.
[0349] 3.4 Thermal Isolation Pockets
[0350] An advantageous arrangement for thermal isolation pockets
for a nucleic acid amplification chamber on a fluidic cartridge
will now be described, which may form an isolated inventive
aspect.
[0351] In nucleic acid amplification and detection, it is
preferable to apply heat evenly throughout the liquid sample.
Whilst it is possible to do this without difficulty in a laboratory
by placing heat sources equidistantly around the sample, it is much
harder to achieve in a cartridge.
[0352] Hence, in one aspect, there is provided a fluidic cartridge
for performing nucleic acid amplification on a liquid sample, the
cartridge comprising at least one sample processing chamber and a
thermally insulating region adjacent the chamber to prevent heat
loss from the chamber through the thermally insulating region.
Preferably the at least one sample processing chamber is one or
both of a nucleic acid amplification chamber and a nucleic acid
detection chamber (hence forth `processing chamber`).
[0353] Preferably the nucleic acid processing chamber is adjacent a
surface (preferably a bottom surface) of the cartridge for
accepting heat from an external source, the chamber situated
between the thermally insulating region and the surface such that
heat passing from the external source through the surface and
thence the chamber is not lost out of the other side of the chamber
owing to the presence of the thermally insulating region. This
arrangement is found to make the change in temperature inside the
chamber (for instance when turning the heat source on and off) as
fast as possible, which is beneficial for performing rapid PCR, for
example.
[0354] This is particularly advantageous because a single heat
source may be placed adjacent the cartridge to supply heat for the
amplification process from one side (the heated side), and yet the
sample within the cartridge will be heated substantially and the
amount of heat lost through the unheated side minimised as far as
possible.
[0355] Preferably the cartridge is comprised of at least a fluidic
layer and a pneumatic layer in contacting arrangement. The nucleic
acid processing chamber may be formed in the fluidic layer and the
thermally insulating region may be formed in the pneumatic layer.
Preferably the fluidic cartridge further comprises a fluidic foil
underneath the fluidic layer, the foil forming the aforementioned
surface for accepting heat. The use of a thin foil maximises the
heat transfer from the external source. The material of the foil
may be chosen to optimise the heat transfer. For instance, a metal
foil may be used, but it is preferred that a polyethylene
terephthalate/polypropylene composite is used due to the advantages
in ease of manufacture of the cartridge, together with material
strength and acceptable heat transfer properties.
[0356] Preferably the thermally insulating region is formed from
one or more sealed thermal isolation pockets formed in the
pneumatic layer and sealed by a pneumatic foil. The pockets may be
filled with gas such as air or may be evacuated during the
manufacturing process such that they provide a vacuum.
[0357] A preferred embodiment of this aspect will now be described
with reference to the exemplary fluidic cartridge. As shown in FIG.
3, the exemplary cartridge 100 comprises, from top to bottom, a
housing 111, a blister sub-assembly 112, a pneumatic foil 113, a
pneumatic layer 114, a fluid layer 115 and a fluidic foil 116.
[0358] Referring to FIGS. 6A and 6B, which shows the pneumatic
layer, six thermally insulating regions 140a-b, 141a-d are
provided. The insulating regions 140a-b are located adjacent two
corresponding amplification chambers formed in the fluidic layer
115, whilst insulating regions 141a-d are located adjacent four
corresponding detection chambers formed in the fluidic layer 115,
when the cartridge is assembled. As shown, the insulating regions
140a-b consist of a plurality of thermal isolation pockets, whereas
insulating regions 141a-d each consist of a single pocket.
[0359] During nucleic acid amplification and detection,
thermocycling of the amplification and detection chambers takes
place. The chambers in the fluidic layer may be heated by applying
heat to the bottom of the cartridge 100, adjacent the fluidic layer
115. The thermal isolation pockets retain the heat within the
cartridge, minimising heat loss from the fluidic layer 115 into the
pneumatic layer 114. The thermal isolation pockets also eliminate
the need for heating of the fluidics cartridge from both the top
and bottom surfaces e.g. heating both the fluidics layer and the
pneumatic layer, simplifying the overall design of the cartridge
and reader.
[0360] The thermal isolation pocket may comprise one large pocket
or multiple smaller pockets. The advantage of using multiple
smaller pockets is that the risk of convection currents being set
up is reduced, thus providing maximal thermal insulation.
[0361] 3.5 Capture Column
[0362] An advantageous arrangement for a filtering device in a
fluidic cartridge (preferably a `capture column`) will now be
described, which may form an isolated inventive aspect.
[0363] Hence, in one aspect, there is provided a fluidic cartridge
comprising a channel through which a liquid sample may pass, the
channel having a filter for capturing biological components and
further comprising: [0364] an upstream portion and a downstream
portion; and [0365] a capture portion between the upstream and
downstream portions in which the filter is arranged; wherein:
[0366] the diameter of the capture portion is greater than the
diameter of the upstream and downstream portions.
[0367] Preferably the capture portion is a chamber within the
channel, the chamber having an inlet surface having an opening
coupled to the upstream portion of the channel and an outlet
surface having an opening coupled to the downstream portion of the
channel.
[0368] Preferably the fluidic cartridge comprises at least two
polymer layers, wherein the upstream portion and an upstream part
of the capture portion of the channel are formed in a first polymer
layer and the downstream portion and a downstream part of the
capture portion of the channel are formed in a second polymer
layer; and wherein the filter is clamped between the first and
second polymer layers.
[0369] Preferably the inlet surface of the chamber comprises
distribution conduits leading radially outwardly from the opening
so as to direct a liquid sample passing through the opening in the
inlet surface radially outwardly.
[0370] Preferably the outlet surface of the chamber comprises
distribution conduits leading radially inwardly toward the opening
so as to direct a liquid sample which has passed through the filter
radially inwardly toward the opening in the outlet surface.
[0371] A preferred embodiment of this aspect will now be described
with reference to the exemplary fluidic cartridge. In the exemplary
cartridge described herein, a capture column 24 is provided along
the main channel (see FIG. 1). As shown in FIGS. 14a and 14b, the
capture column 24 has filter 340 which binds DNA from lysed
material before releasing it during elution. As shown in FIG. 14a,
capture column 24 comprises an inlet channel 342 leading into a
capture chamber 344 at an upstream end 346, and an outlet channel
350 leading from capture chamber 344 at a downstream end 348.
[0372] A filter 340 is provided in chamber 344, perpendicular to
the direction of flow of fluid through the main channel, such that
fluid must pass through filter 340 when passing from the upstream
end of the main channel 342 to the downstream end 350 of the main
channel.
[0373] Referring now to FIG. 14b, the inlet and outlet walls (only
one is shown) of the chamber comprise distribution conduits 352
configured to direct fluid radially outwardly into the chamber 344
as it enters the chamber, and radially inwardly toward the exit
aperture after it has passed through the filter 340.
[0374] 3.6 Waste Chamber
[0375] An advantageous arrangement for waste chamber in a fluidic
cartridge will now be described, which may form an isolated
inventive aspect.
[0376] Hence, in one aspect, there is provided a fluidic cartridge
comprising a channel through which a liquid sample may pass and a
waste chamber for receiving fluid from the channel, the waste
chamber comprising: [0377] a pipe, coupled to the channel,
extending from a bottom surface of the waste chamber and having an
opening elevated above the bottom surface to pass fluid from the
channel into the chamber; and [0378] a vent within the waste
chamber configured to vent the waste chamber to atmosphere.
[0379] Preferably the vent comprises a second pipe, coupled to a
vent channel within the cartridge, extending from the bottom
surface of the waste chamber and having an opening elevated above
the bottom surface. Preferably the vent passageway comprises at
least one Anderson impactor.
[0380] Preferably at least one absorbent pad is provided within the
waste chamber.
[0381] A preferred embodiment of this aspect will now be described
with reference to the exemplary fluidic cartridge. In the exemplary
cartridge described herein, a waste chamber is provided for
collecting and storing waste fluid which is produced during washing
etc. Waste chamber 10 is shown in more detail in FIGS. 15a and 15b.
Waste chamber 38 comprises a pipe 360, extending substantially
vertically from a bottom surface 362 of waste chamber 38. The pipe
38 defines a channel having a first end 364 connected to the bottom
surface of the waste chamber 38 and fluidly connected to the main
channel 16. A second end 366 of fluid pipe 360 is disposed within
waste chamber 38, and has an opening through which fluid can flow
into the waste chamber.
[0382] Preferably, pipe 360 is substantially vertical, and
perpendicular to the bottom surface of the waste chamber 38. The
opening at the second end of pipe 360 is located near the top of
the waste chamber 38 as shown in FIG. 15b. By providing the first
opening near the top of the waste chamber, the risk of leakage is
minimised should the cartridge be turned upside down.
[0383] Absorbent pads 368 are also provided in the waste chamber.
Preferably, the upper surface of absorbent pads 368 should also be
near the top of waste chamber 38, even more preferably, the top of
absorbent pads 368 should be substantially level with the opening
at the second end 366.
[0384] In the exemplary cartridge described herein, a second
opening 370 is provided in waste chamber 38 as shown in FIG. 15b.
The second opening 370 is configured to vent main channel 16 via
waste chamber 28 to atmospheric pressure. This avoids putting a
back pressure along the main channel as the waste channel fills
with fluid. Preferably, the second opening 370 is provided at the
end of a second pipe 372 protruding from the bottom surface of
waste chamber 38. The second opening 370 may be fluidly connected
to a vent passageway (not shown) which has an opening outside of
the cartridge housing to allow the waste chamber to remain at
atmospheric pressure. However, venting the waste chamber outside
the cartridge carries a small risk of aerosol contamination. To
reduce this, the vent path has impact traps and vents under the
cartridge cover.
[0385] The skilled person will be capable of modifying the
exemplary cartridge to implement the inventive aspects described
herein in various ways depending on circumstances. It is intended
that the scope of the present invention is defined by the following
claims.
* * * * *